Compositions, methods and kits to detect herpes simplex virus nucleic acids

ABSTRACT

The disclosed invention is related to methods, compositions, kits and isolated nucleic acid sequences for targeting Herpes Simplex Virus (HSV) nucleic acid (e.g., HSV-1 and/or HSV-2 nucleic acid). Compositions include amplification oligomers, detection probe oligomers and/or target capture oligomers. Kits and methods comprise at least one of these oligomers.

PRIORITY

This application is a Divisional of U.S. patent application Ser. No.14/395,039, filed Oct. 16, 2014, which is a 371 of PCT/US2013/037808,filed Apr. 23, 2013, which claims the benefit of priority to thefollowing applications: U.S. Provisional Application No. 61/637,769filed 24 Apr.2012; U.S. Provisional Application No. 61/748,854 filed 04Jan. 2013; and U.S. Provisional Application No. 61/773,718 filed 06 Mar.2013. The entire contents of each of these priority documents areincorporated herein by reference.

FIELD

The present invention relates to the detection of infectious agents,more specifically to the detection of Herpes Simplex virus (HSV).Compositions, methods and kits are described for the detection of HSV(including HSV types 1 and 2) by using in vitro nucleic acidamplification techniques.

BACKGROUND

Herpes simplex virus (HSV) is part of the larger herpes virus family,including Varicella-Zoster virus (VZV), Epstein-Barr virus (EBV) andCytomegalovirus (CMV). It is an enveloped double-stranded DNA viruscausing infections in humans. HSV is classified into various types,including HSV-1 and HSV-2. The complete genomes of human HSV-1 and HSV-2have been sequenced (see, e.g., NCBI Accession Nos.NC_001806.1/GI:9629378 and NC_001798.1/GI:9629267, respectively; seealso accession numbers X14112 and Z86099, respectively). Both HSV-1 andHSV-2 can cause disease in humans and exposure or infection is fairlycommon in adult populations. Up to 80% of the U.S. adult population hasbeen exposed to HSV-1 and approximately 20% of the U.S. population hascontracted HSV-2 infections.

HSV infection symptoms include the common cold sore found near the lipsand also genital herpes. The virus can also cause keratoconjunctivitis,with the potential to lead to blindness, and encephalitis. Oncesubsided, the virus remains in a latent state inside nerve cells(ganglia) that supply nerve fibres to the infected area. The virus canbecome reactivated and travels through the nerve fibres back to theskin, thereby causing recurrent disease.

HSV-2 is commonly associated with newborn encephalitis where it isassociated with maternal genital infections. HSV-related encephalitishas the highest fatality rate of all types of encephalitis with anannual incidence of 1 to 4 per million HSV encephalitis affects peopleof all ages and at any time of the year. In adults, HSV-relatedencephalitis is thought to be due to a reactivation of a latent virus.Symptoms may include fever, headaches, seizures, an altered level ofconsciousness and personality changes. The similarity of these symptomsto other maladies makes clinical diagnosis difficult. If left untreated,the mortality rate for Herpes Simplex Encephalitis (HSE) is as high asseventy percent, compared with as low as nineteen percent among thosewho receive treatment. Of the treated patients, about one third canreturn to normal function.

One mechanism for transmission of HSV is by sexual transmission. Thisroute of transmission presents a serious consequence of HSV infection inthe transmission of the HIV virus. HIV transmission is five times morelikely to occur from an HIV/HSV-2-coinfected person with genitalulceration and HIV acquisition is twice as likely in someonesero-positive for HSV-2.

Accurate diagnosis of HSV infection is essential if transmission ratesof HSV and its consequences are to be reduced. Although it is notpossible to eradicate HSVs from an infected individual, episodictreatment with nucleoside analogue drugs will shorten the duration ofthe clinical episode and can also reduce the risk of transmission of thevirus when continuously administered as daily suppressive therapy.Clinical diagnosis of HSV infection has been reported to have a poorsensitivity of only approximately 40% (Expert Rev. Mol. Diagn. 4,485-493 (2004); Sex. Trans. Dis. 17, 90-94 (1990)) so rapid reliabletests with good sensitivity and specificity are needed to improvediagnostic accuracy in those with and without symptoms. Tests are alsorequired that differentiate between HSV-1 and -2.

Current diagnostic methods for HSV include viral culture, serologicaltests and nucleic acid amplification testing (NAAT).

Culture and typing were once considered the gold standard for diagnosisbut its usefulness is severely limited by the stage of clinical disease.When testing early vesicular lesions, the culture detection rate isabout 90% whereas in older crusted lesions this falls to only 27%(Genitourin. Med. 64, 103-106 (1988)). Another problem with this methodis that it is slow since it takes 3 days for the majority of cultureisolates to appear positive. The liability of the virus also means thatsamples must be transported rapidly with maintenance of the cold chainotherwise much reduced sensitivity will result due to, for example,bacterial outgrowth.

Detection of HSV infections has improved dramatically with the advent oftype-specific HSV antibody serology testing (Am. J. Clin. Pathol. 120,829-844 (2003). These tests are sensitive and can distinguish betweenHSV-1 and HSV-2 antibodies. However, type specific antibody tests sufferfrom false positive results and are also considered inadequate due to adelay of between two and three weeks in appearance of antibody responseafter initial infection. The performance of the same test can also vary,giving different sensitivities and specificities depending on thepopulation tested (Clin. Microbiol. Infect. 10, 530-536 (2004)). Forthese reasons, they are not considered suitable for general populationscreening.

NAAT testing for HSV provides for the direct detection of viral DNA fromspecimens by amplifying DNA sequences using HSV-1 or -2 specific primersand has been shown to be superior to culture (Sex. Trans. Infect. 78,21-25 (2002); Sex. Trans. Infect. 80, 406-410 (2004)) and highlyspecific as compared to cell culture (J. Infect. Dis. 1345-1351(2003)).Different HSV genes have been identified as targets for DNAamplification, among them, DNA polymerase glycoprotein. NAAT basedtesting for HSV has utilised Strand-displacement amplification (SDA),PCR, real time PCR and the TaqMan® PCR detection system. NAAT basedassays for HSV are now considered to be the gold standard. However,PCR-based amplification assays are not without their limitations. Forexample, tests may take up to 2 days to complete and require specializedthermo-cycling equipment.

Sciortino et al. (2001) J. Virol. 75, 17 pp. 8105-8116 describe a methodfor the detection of HSV using reverse transcribed RNAs that weredetected by PCR. A set of 90 primers were designed to amplify all of the84 expressed ORFs of HSV. One primer pair was designed to amplify aportion of the UL42 ORF of HSV-1, hybridising to regions 301 to 322 and680 to 701 of GenBank Accession No: GU734771.1, GI:290766003, region92815 . . . 94534. However, the method described therein suffers fromthe problems associated with PCR-based amplification methodologies andalso requires a reverse transcription step which adds yet furthercomplexity to the method. It is also believed that this assay would notbe able to discriminate between HSV-1 and HSV-2 nucleic acids.

A need remains for a diagnostic test that provides sensitive andspecific detection of HSV in a relatively short time so that infectedindividuals may be treated promptly to limit morbidity and prevent thespread of infection. A test of this kind that distinguishes betweenHSV-1 and/or HSV-2 would also be desirable and so a type determinationof HSV that is present in the sample can be made.

SUMMARY

The present invention relates to methods, compositions, kits and nucleicacids for determining the presence of HSV, specifically HSV-1 and/orHSV-2, in a sample. The methods involve the amplification of viralnucleic acid to detect the HSV target sequence in the sample. Themethods can advantageously provide for the sensitive detection andtype-determination of HSV. The present invention is also directed to amethod—such as a TMA based method—for the detection of HSV whichprovides for the direct, rapid, specific and sensitive detection of HSVRNA. Targeting single stranded RNA is beneficial over targeting thedouble stranded genomic DNA because there is no need for an additionaldenaturation step which otherwise adds further complexity to the method.The use of RNA can also provide improved amplification oligomerefficiency when methods—such as TMA—start from a single stranded nucleicacid molecule. A distinct viral RNA expressed in infected cells andpackaged by HSV-1 and HSV-2 virions, UL42, was selected as a target foramplification and detection. (Georgopoulou, J. Virol. 67, 3961, (1993);McGeoch, J. Gen. Virol. 69, 1531, (1988); Sciortino et al. PNAS 99, 12,8318, (2002); and Sciortino, J. Virol. 75, 8105, (2001)).

A viral nucleic acid that is targeted according to the present inventionis the UL42 open reading frame (ORF) of HSV. This ORF is present in bothHSV-1 and HSV-2. The nucleic acid sequence of the UL42 ORF in HSV-1 isdifferent than the UL42 ORF nucleic acid sequence in HSV-2. Thisdifference in nucleic acid sequences can be exploited by designingamplification oligomers and/or nucleic acid probes that are specific foreach of the sequences. Thus, the methods of the present invention can beused to distinguish between the two types of HSV. Accordingly, it ispossible to determine if a sample comprises HSV-1 or HSV-2 or acombination thereof. Accordingly, it is possible to determine if asample comprises HSV-1 or HSV-2 or a combination thereof in both earlyand late stages of the viral lifecycle.

DNA sequences encoding the UL42 ORF from HSV-1 (SEQ ID NO:1) and HSV-2(SEQ ID NO:2) are shown in Table 17. FIGS. 1 and 2 (illustrated as FIG.1A, FIG. 1B, FIG. 1C, FIG. 2A, and FIG. 2B) further illustrate the UL42ORF from HSV-1 and HSV-2, respectively. Methods herein target the RNAsequences of SEQ ID NO:1 and 2. Methods herein may also target the DNAsequence of SEQ ID NO:1. Methods herein may also target the DNA sequenceof SEQ ID NO:2.

In one aspect, the present invention provides a method for determiningthe presence or absence of Herpes Simplex Virus 1 (HSV-1) in a sample.The method includes the step of (1) contacting a sample, suspected ofcontaining HSV-1, with at least two oligomers for amplifying a targetregion of an HSV-1 target nucleic acid, where the at least twoamplification oligomers include (a) a first amplification oligomercomprising a first target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides contained in the sequence of SEQ IDNO:31 and that includes at least the sequence of SEQ ID NO:30, and (b) asecond amplification oligomer comprising a second target-hybridizingsequence that is from about 15 to about 27 contiguous nucleotidescontained in the sequence of SEQ ID NO:33 and that includes at least thesequence of SEQ ID NO:32. The method further includes (2) performing anin vitro nucleic acid amplification reaction, where any HSV-1 targetnucleic acid present in the sample is used as a template for generatingan amplification product, and (3) detecting the presence or absence ofthe amplification product, thereby indicating the presence or absence ofHSV-1 in the sample. In some variations, the first target-hybridizingsequence is contained in the sequence of SEQ ID NO:29 and/or includes atleast the sequence of SEQ ID NO:28. Suitable first target-hybridizingsequences for the first amplification oligomer include SEQ ID NO:20, SEQID NO:6, and SEQ ID NO:12. Suitable second target-hybridizing sequencesfor the second amplification oligomer include SEQ ID NO:7 and SEQ IDNO:9. In more particular variations, the first and secondtarget-hybridizing sequences respectively have the nucleotide sequencesof (i) SEQ ID NO:20 and SEQ ID NO:7, (ii) SEQ ID NO:6 and SEQ ID NO:7,or (iii) SEQ ID NO:6 and SEQ ID NO:9.

In some embodiments of a method for determing the presence or absence ofHSV-1, the first amplification oligomer is a promoter primer or promoterprovider further comprising a promoter sequence located 5′ to the firsttarget-hybridizing sequence. A particularly suitable promoter sequenceis a T7 promoter sequence such as, e.g., the nucleotide sequence of SEQID NO:54. In some such variations, the first amplification oligomer hasa sequence selected from SEQ ID NO:19, SEQ ID NO:5, and SEQ ID NO:11.

In certain embodiments, the detecting step (3) includes contacting thein vitro nucleic acid amplification reaction with a detection probeoligomer configured to specifically hybridize to the amplificationproduct under conditions whereby the presence or absence of theamplification product is determined, thereby indicating the presence orabsence of HSV-1 in the sample. Typically, the detection probe oligomerincludes a target-hybridizing sequence that is from about 14 to about 40nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:1 from about nucleotideposition 635 to about nucleotide position 683. For example, thedetection probe target-hybridizing sequence may be contained in thesequence of SEQ ID NO:40 or SEQ ID NO:41 and include at least thesequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:38.

In some embodiments of a detection probe target-hybridizing sequencethat includes at least the sequence of SEQ ID NO:34 or SEQ ID NO:35, thetarget-hybridizing sequence is contained in the sequence of SEQ ID NO:36or SEQ ID NO:37. In specific variations, the detection probetarget-hybridizing sequence is SEQ ID NO:8 or SEQ ID NO:22; in some suchvariations, the first and second amplification oligomertarget-hybridizing sequences respectively have the nucleotide sequencesof (i) SEQ ID NO:20 and SEQ ID NO:7, (ii) SEQ ID NO:6 and SEQ ID NO:7,or (iii) SEQ ID NO:6 and SEQ ID NO:9.

In some embodiments of a detection probe target-hybridizing sequencethat includes at least the sequence of SEQ ID NO:38, the detection probetarget-hybridizing sequence is contained in the sequence of SEQ IDNO:39. In specific variations, the detection probe target-hybridizingsequence has the sequence of SEQ ID NO:10; in some such variations, thefirst and second amplification oligomer target-hybridizing sequencesrespectively have the nucleotide sequences of (i) SEQ ID NO:20 and SEQID NO:7, (ii) SEQ ID NO:6 and SEQ ID NO:7, or (iii) SEQ ID NO:6 and SEQID NO:9.

In one aspect, the present invention provides a method for determiningthe presence or absence of Herpes Simplex Virus 2 (HSV-2) in a sample.The method includes the step of (1) contacting a sample, suspected ofcontaining HSV-2, with at least two oligomers for amplifying a targetregion of an HSV-2 target nucleic acid, where the at least twoamplification oligomers include (a) a first amplification oligomercomprising a first target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides (i) contained in the sequence of SEQ IDNO:49 and that includes at least the sequence of SEQ ID NO:48 or (ii)contained in the sequence of SEQ ID NO:43 and that includes at least thesequence of SEQ ID NO:42; and (b) a second amplification oligomercomprising a second target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides (i) contained in the sequence of SEQ IDNO:51 and that includes at least the sequence of SEQ ID NO:50 or (ii)contained in the sequence of SEQ ID NO:45 and that includes at least thesequence of SEQ ID NO:44. The method further includes (2) performing anin vitro nucleic acid amplification reaction, where any HSV-2 targetnucleic acid present in the sample is used as a template for generatingan amplification product, and (3) detecting the presence or absence ofthe amplification product, thereby indicating the presence or absence ofHSV-2 in the sample. In some embodiments, the first target hybridizingsequence is contained in the sequence of SEQ ID NO:49 and includes atleast the sequence of SEQ ID NO:48, and the second target hybridizingsequence is contained in the sequence of SEQ ID NO:51 and includes atleast the sequence of SEQ ID NO:50. In particular variations, the firsttarget-hybridizing sequence has the sequence of SEQ ID NO:24 and/or thesecond target hybridizing sequence has the sequence of SEQ ID NO:25. Inother embodiments, the first target hybridizing sequence is contained inthe sequence of SEQ ID NO:43 and includes at least the sequence of SEQID NO:42, and the second target hybridizing sequence is contained in thesequence of SEQ ID NO:45 and includes at least the sequence of SEQ IDNO:44. In particular variations, the first target-hybridizing sequencehas the sequence of SEQ ID NO:14 and/or the second target hybridizingsequence has the sequence of SEQ ID NO:15.

In some embodiments of a method for determing the presence or absence ofHSV-2, the first amplification oligomer is a promoter primer or promoterprovider further comprising a promoter sequence located 5′ to the firsttarget-hybridizing sequence. A particularly suitable promoter sequenceis a T7 promoter sequence such as, e.g., the nucleotide sequence of SEQID NO:54. In some such variations, the first amplification oligomer hasa sequence selected from SEQ ID NO:23 and SEQ ID NO:13.

In certain embodiments of a method for determining the presence orabsence of HSV-2, the detecting step (3) includes contacting the invitro nucleic acid amplification reaction with a detection probeoligomer configured to specifically hybridize to the amplificationproduct under conditions whereby the presence or absence of theamplification product is determined, thereby indicating the presence orabsence of HSV-2 in the sample. In some embodiments—where the firsttarget hybridizing sequence is contained in the sequence of SEQ ID NO:49and includes at least the sequence of SEQ ID NO:48, and the secondtarget hybridizing sequence is contained in the sequence of SEQ ID NO:51and includes at least the sequence of SEQ ID NO:50—the detection probeoligomer includes a target-hybridizing sequence that is from about 14 toabout 25 nucleotides in length and configured to specifically hybridizeto a target sequence contained within SEQ ID NO:2 from about nucleotideposition 608 to about nucleotide position 632. In certain variations,the detection probe target-hybridizing sequence is contained in thesequence of SEQ ID NO:53 and includes at least the sequence of SEQ IDNO:52. A particularly suitable detection probe target-hybridizingsequence has the sequence of SEQ ID NO:27; in some such variations, thefirst and second amplification oligomer target-hybridizing sequenceshave the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25,respectively.

In other embodiments of a method for determining the presence or absenceof HSV-2 comprising the use of a detection probe—where the first targethybridizing sequence is contained in the sequence of SEQ ID NO:43 andincludes at least the sequence of SEQ ID NO:42, and the second targethybridizing sequence is contained in the sequence of SEQ ID NO:45 andincludes at least the sequence of SEQ ID NO:44—the detection probeoligomer includes a target-hybridizing sequence that is from about 14 toabout 30 nucleotides in length and configured to specifically hybridizeto a target sequence contained within SEQ ID NO:2 from about nucleotideposition 549 to about nucleotide position 578. In certain variations,the detection probe target-hybridizing sequence is contained in thesequence of SEQ ID NO:47 and includes at least the sequence of SEQ IDNO:46. A particularly suitable detection probe target-hybridizingsequence has the sequence of SEQ ID NO:16; in some such variations, thefirst and second amplification oligomer target-hybridizing sequenceshave the nucleotide sequences of SEQ ID NO:14 and SEQ ID NO:15,respectively.

Typically, a method for determining the presence or absence of HSV-1 orHSV-2 as above further includes purifying the HSV-1 or HSV-2 targetnucleic acid from other components in the sample before step (1). Inparticular embodiments, the purifying step includes contacting thesample with at least one capture probe oligomer comprising atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe. Suitable target-hybridizingsequences include SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:70, and SEQ IDNO:72. In more particular variations, the capture probe oligomer has asequence selected from SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:69, and SEQID NO:71.

In certain embodiments in which the purifying step includes contactingthe sample with at least one capture probe oligomer comprising atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe, the target-hybridizing sequence isfrom about 15 to about 30 contiguous nucleotides contained in thesequence of SEQ ID NO:76 and includes at least the sequence of SEQ IDNO:75. In some variations, the capture probe target-hybridizing sequenceis contained in the segue nce of SEQ ID NO:74 and/or includes at leastthe sequence of SEQ ID NO:75. Particularly suitable target-hybridizingsequences include SEQ ID NO:70 and SEQ ID NO:72. In some embodiments,the purifying step further includes contacting the sample with a secondcapture probe oligomer comprising a target-hybridizing sequenceconfigured to specifically hybridize to the HSV-1 and/or HSV-2 targetnucleic acid, where the second capture probe target-hybridizing sequenceis covalently attached to a sequence or moiety that binds to animmobilized probe; in some such variations, the second capture probeoligomer has a target-hybridizing sequence as shown in SEQ ID NO:4 orSEQ ID NO:18.

In some embodiments of a method as above for determining the presence orabsence of HSV-1 or HSV-2 utilizing a detection probe oligomer, thedetection probe includes at least one label. In specific variations, thelabel is a chemiluminescent label or a fluorescent label. In someembodiments utilizing a labeled detection probe, the detecting step (3)occurs during the amplifying step (2). Particularly suitable detectionprobes that may comprise a fluorescent label and a quencher include amolecular torch, a molecular beacon, and a TaqMan detection probe.

In still other embodiments of a method utilizing a detection probeoligomer, the detection probe further includes a non-target-hybridizingsequence. In particular embodiments, a detection probe comprising anon-target-hybridizing sequence is a hairpin detection probe such as,e.g., a molecular torch or a molecular beacon.

In certain embodiments of a method for determining the presence orabsence of HSV-1 or HSV-2 as above, the amplification reaction at step(2) is an isothermal amplification reaction or a PCR amplificationreaction. In specific variations, the isothermal amplification reactionis a transcription-mediated amplification (TMA) reaction. In someembodiments of a method utilizing an isothermal or PCR amplificationreaction, the reaction is a real-time amplification reaction.

In another aspect, the present invention provides a combination of atleast two oligomers for determining the presence or absence of HerpesSimplex Virus 1 (HSV-1) in a sample. The oligomer combination includesfirst and second amplification oligomers for amplifying a target regionof an HSV-1 target nucleic acid, where (a) the first amplificationoligomer comprises a first target-hybridizing sequence that is fromabout 15 to about 27 contiguous nucleotides contained in the sequence ofSEQ ID NO:31 and that includes at least the sequence of SEQ ID NO:30,and (b) the second amplification oligomer comprises a secondtarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides contained in the sequence of SEQ ID NO:33 and that includesat least the sequence of SEQ ID NO:32. In some variations, the firsttarget-hybridizing sequence is contained in the sequence of SEQ ID NO:29and/or includes at least the sequence of SEQ ID NO:28. Suitable firsttarget-hybridizing sequences for the first amplification oligomerinclude SEQ ID NO:20, SEQ ID NO:6, and SEQ ID NO:12. Suitable secondtarget-hybridizing sequences for the second amplification oligomerinclude SEQ ID NO:7 and SEQ ID NO:9. In more particular variations, thefirst and second target-hybridizing sequences respectively have thenucleotide sequences of (i) SEQ ID NO:20 and SEQ ID NO:7, (ii) SEQ IDNO:6 and SEQ ID NO:7, or (iii) SEQ ID NO:6 and SEQ ID NO:9.

In some embodiments of an oligomer combination for determing thepresence or absence of HSV-1, the first amplification oligomer is apromoter primer or promoter provider further comprising a promotersequence located 5′ to the first target-hybridizing sequence. Aparticularly suitable promoter sequence is a T7 promoter sequence suchas, e.g., the nucleotide sequence of SEQ ID NO:54. In some suchvariations, the first amplification oligomer has a sequence selectedfrom SEQ ID NO:19, SEQ ID NO:5, and SEQ ID NO:11.

In certain embodiments, an oligomer combination for determining thepresence or absence of HSV-1 as above further includes a detection probeoligomer. Typically, the detection probe oligomer includes atarget-hybridizing sequence that is from about 14 to about 40nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:1 from about nucleotideposition 635 to about nucleotide position 683. For example, thedetection probe target-hybridizing sequence may be contained in thesequence of SEQ ID NO:40 or SEQ ID NO:41 and include at least thesequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:38.

In some embodiments of a detection probe target-hybridizing sequencethat includes at least the sequence of SEQ ID NO:34 or SEQ ID NO:35, thetarget-hybridizing sequence is contained in the sequence of SEQ ID NO:36or SEQ ID NO:37. In specific variations, the detection probetarget-hybridizing sequence is SEQ ID NO:8 or SEQ ID NO:22; in some suchvariations, the first and second amplification oligomertarget-hybridizing sequences respectively have the nucleotide sequencesof (i) SEQ ID NO:20 and SEQ ID NO:7, (ii) SEQ ID NO:6 and SEQ ID NO:7,or (iii) SEQ ID NO:6 and SEQ ID NO:9.

In some embodiments of a detection probe target-hybridizing sequencethat includes at least the sequence of SEQ ID NO:38, the detection probetarget-hybridizing sequence is contained in the sequence of SEQ IDNO:39. In specific variations, the detection probe target-hybridizingsequence has the sequence of SEQ ID NO:10; in some such variations, thefirst and second amplification oligomer target-hybridizing sequencesrespectively have the nucleotide sequences of (i) SEQ ID NO:20 and SEQID NO:7, (ii) SEQ ID NO:6 and SEQ ID NO:7, or (iii) SEQ ID NO:9 and SEQID NO:9.

In another aspect, the present invention provides a combination of atleast two oligomers for determining the presence or absence of HerpesSimplex Virus 2 (HSV-2) in a sample. The oligomer combination includesfirst and second amplification oligomers for amplifying a target regionof an HSV-2 target nucleic acid, where (a) a first amplificationoligomer comprising a first target-hybridizing sequence that is fromabout 15 to about 27 contiguous nucleotides (i) contained in thesequence of SEQ ID NO:49 and that includes at least the sequence of SEQID NO:48 or (ii) contained in the sequence of SEQ ID NO:43 and thatincludes at least the sequence of SEQ ID NO:42; and (b) a secondamplification oligomer comprising a second target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides (i) containedin the sequence of SEQ ID NO:51 and that includes at least the sequenceof SEQ ID NO:50 or (ii) contained in the sequence of SEQ ID NO:45 andthat includes at least the sequence of SEQ ID NO:44. In someembodiments, the first target hybridizing sequence is contained in thesequence of SEQ ID NO:49 and includes at least the sequence of SEQ IDNO:48, and the second target hybridizing sequence is contained in thesequence of SEQ ID NO:51 and includes at least the sequence of SEQ IDNO:50. In particular variations, the first target-hybridizing sequencehas the sequence of SEQ ID NO:24 and/or the second target hybridizingsequence has the sequence of SEQ ID NO:25. In other embodiments, thefirst target hybridizing sequence is contained in the sequence of SEQ IDNO:43 and includes at least the sequence of SEQ ID NO:42, and the secondtarget hybridizing sequence is contained in the sequence of SEQ ID NO:45and includes at least the sequence of SEQ ID NO:44. In particularvariations, the first target-hybridizing sequence has the sequence ofSEQ ID NO:14 and/or the second target hybridizing sequence has thesequence of SEQ ID NO:15.

In some embodiments of an oligomer combination for determing thepresence or absence of HSV-2, the first amplification oligomer is apromoter primer or promoter provider further comprising a promotersequence located 5′ to the first target-hybridizing sequence. Aparticularly suitable promoter sequence is a T7 promoter sequence suchas, e.g., the nucleotide sequence of SEQ ID NO:54. In some suchvariations, the first amplification oligomer has a sequence selectedfrom SEQ ID NO:23 and SEQ ID NO:13.

In certain embodiments, an oligomer combination for determining thepresence or absence of HSV-2 as above further includes a detection probeoligomer. In some embodiments—where the first target hybridizingsequence is contained in the sequence of SEQ ID NO:49 and includes atleast the sequence of SEQ ID NO:48, and the second target hybridizingsequence is contained in the sequence of SEQ ID NO:51 and includes atleast the sequence of SEQ ID NO:50—the detection probe oligomer includesa target-hybridizing sequence that is from about 14 to about 25nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:2 from about nucleotideposition 608 to about nucleotide position 632. In certain variations,the detection probe target-hybridizing sequence is contained in thesequence of SEQ ID NO:53 and includes at least the sequence of SEQ IDNO:52. A particularly suitable detection probe target-hybridizingsequence has the sequence of SEQ ID NO:27; in some such variations, thefirst and second amplification oligomer target-hybridizing sequenceshave the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25,respectively.

In other embodiments of an oligomer combination for determining thepresence or absence of HSV-2 comprising a detection probe—where thefirst target hybridizing sequence is contained in the sequence of SEQ IDNO:43 and includes at least the sequence of SEQ ID NO:42, and the secondtarget hybridizing sequence is contained in the sequence of SEQ ID NO:45and includes at least the sequence of SEQ ID NO:44—the detection probeoligomer includes a target-hybridizing sequence that is from about 14 toabout 30 nucleotides in length and configured to specifically hybridizeto a target sequence contained within SEQ ID NO:2 from about nucleotideposition 549 to about nucleotide position 578. In certain variations,the detection probe target-hybridizing sequence is contained in thesequence of SEQ ID NO:47 and includes at least the sequence of SEQ IDNO:46. A particularly suitable detection probe target-hybridizingsequence has the sequence of SEQ ID NO:16; in some such variations, thefirst and second amplification oligomer target-hybridizing sequenceshave the nucleotide sequences of SEQ ID NO:14 and SEQ ID NO:15,respectively.

An oligomer combination for determining the presence or absence of HSV-1or HSV-2 as above may also include at least one capture probe oligomer.In some such embodiments, the capture probe oligomer includes atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe. Suitable target-hybridizingsequences include SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:70, and SEQ IDNO:72. In more particular variations, the capture probe oligomer has asequence selected from SEQ ID NO:3, SEQ ID NO:17, SEQ ID NO:69, and SEQID NO:71.

In certain embodiments in which the oligomer combination includes atleast one capture probe oligomer comprising a target-hybridizingsequence covalently attached to a sequence or moiety that binds to animmobilized probe, the target-hybridizing sequence is from about 15 toabout 30 contiguous nucleotides contained in the sequence of SEQ IDNO:76 and includes at least the sequence of SEQ ID NO:75. In somevariations, the capture probe target-hybridizing sequence is containedin the sequence of SEQ ID NO:74 and/or includes at least the sequence ofSEQ ID NO:75. Particularly suitable target-hybridizing sequences includeSEQ ID NO:70 and SEQ ID NO:72. In some embodiments, the oligomercombination includes a second capture probe oligomer comprising atarget-hybridizing sequence configured to specifically hybridize to theHSV-1 and/or HSV-2 target nucleic acid, where the second capture probetarget-hybridizing sequence is covalently attached to a sequence ormoiety that binds to an immobilized probe; in some such variations, thesecond capture probe oligomer has a target-hybridizing sequence as shownin SEQ ID NO:4 or SEQ ID NO:18.

In some embodiments of an oligomer combination as above for determiningthe presence or absence of HSV-1 or HSV-2 and comprising a detectionprobe oligomer, the detection probe includes at least one label. Inspecific variations, the label is a chemiluminescent label or afluorescent label. In some embodiments, the detection probe includes afluorescent label and a quencher. Particularly suitable detection probesthat may comprise a fluorescent label and a quencher include a moleculartorch, a molecular beacon, and a TaqMan detection probe.

In still other embodiments of an oligomer combination comprising adetection probe oligomer, the detection probe further includes anon-target-hybridizing sequence. In particular embodiments, a detectionprobe comprising a non-target-hybridizing sequence is a hairpindetection probe such as, e.g., a molecular torch or a molecular beacon.

In another aspect, the present invention provides a capture probeoligomer for isolating at least one of a HSV-1 target nucleic acid andHSV-2 target nucleic acid from a sample. In certain embodiments, thecapture probe oligomer comprises a target-hybridizing sequencecovalently attached to a sequence or moiety that binds to an immobilizedprobe, where the target-hybridizing sequence is from about 15 to about30 contiguous nucleotides contained in the sequence of SEQ ID NO:76 andincludes at least the sequence of SEQ ID NO:75. In some variations, thecapture probe target-hybridizing sequence is contained in the sequenceof SEQ ID NO:74 and/or includes at least the sequence of SEQ ID NO:75.Particularly suitable target-hybridizing sequences include SEQ ID NO:70and SEQ ID NO:72.

In yet another aspect, the present invention provides a combination ofat at least two oligomers for isolating at least one of a HSV-1 targetnucleic acid and a HSV-2 target nucleic acid from a sample. In certainembodiments, the oligomer combination includes (1) a first capture probeoligomer comprising a first target-hybridizing sequence that is fromabout 15 to about 30 contiguous nucleotides contained in the sequence ofSEQ ID NO:76 and includes at least the sequence of SEQ ID NO:75, and (2)a second capture probe oligomer comprising a second target-hybridizingsequence configured to specifically hybridize to at least one of theHSV-1 and HSV-2 target nucleic acids. Typically, each of the first andsecond target-hybridizing sequences is covalently attached to a sequenceor moiety that binds to an immobilized probe. In some variations, thefirst capture probe target-hybridizing sequence is contained in thesequence of SEQ ID NO:74 and/or includes at least the sequence of SEQ IDNO:75. Particularly suitable first target-hybridizing sequences includeSEQ ID NO:70 and SEQ ID NO:72. In some embodiments, the second captureprobe target-hybridizing sequence is a sequence as shown in SEQ ID NO:4or SEQ ID NO:18.

In other aspects, the present invention provides a kit or a reactionmixture comprising an oligomer combination as above.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (illustrated as FIG. 1A, FIG. 1B, & FIG. 1C) illustrates areference sequence for the UL42 gene from a representative HSV-1sequence (SEQ ID NO:1). Nucleotide positions 92,815-94,534 of GenBankAccession No. GU734771.1 (GI:290766003) are shown.(>gb|GU734771.1|:92815-94534 Human herpesvirus 1 strain F, completegenome).

FIG. 2 (illustrated as FIG. 2A, & FIG. 2B)_illustrates a referencesequence for the UL42 gene from a representative HSV-2 sequence (SEQ IDNO:2). Nucleotide positions 93,769-95,181 of GenBank Accession No.Z86099.2 (GI:6572414) are shown. (>gi|6572414:93769-95181 Herpes simplexvirus type 2 (strain HG52), complete genome).

DETAILED DESCRIPTION

I. Overview

Nucleic acid oligomer sequences are disclosed that may serve asamplification oligomers for amplification of HSV nucleic acids,including HSV-1 and/or HSV-2 nucleic acids. An HSV nucleic acid may bedetected in a sample by using a method of in vitro nucleic acidamplification, preferably by using a transcription-mediatedamplification reaction such as TMA or NASBA, and detection of anamplified nucleic acid sequence, optionally using a detection probe. Adetection probe hybridizes specifically to a portion of the amplifiedviral sequence, either after completion of or during the amplificationprocess. In one embodiment, the detection probes hybridizes specificallyto a portion of the amplified HSV-1 or HSV-2 sequence, either aftercompletion of or during the amplification process. In particularvariations, a detection probe is able to discriminate between HSV-1 andHSV-2 nucleic acids and so it is possible to determine if either HSV-1and/or HSV-2 nucleic acid is present in the sample under test. Someembodiments detect the amplified products by using a homogeneousdetection method that detects, in a mixture, a labeled probe boundspecifically to an amplified sequence (see, e.g., Arnold et al., 1989,Clin. Chem. 35:1588-1594; U.S. Pat. No. 5,658,737, Nelson et al., andU.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al.). Embodiments ofthe methods also use oligonucleotide sequences that serve as captureprobes for processing a sample to capture the target HSV nucleic acidand separate it from other sample components (see, e.g., U.S. Pat. Nos.6,110,678, 6,280,952 and 6,534,273).

Methods disclosed herein can be used to detect HSV nucleic acids presentin samples from or derived from animals and humans, preferably frombiopsies of genital lesions, anogenital lesions, oral lesions,mucocutanoeus lesions, skin lesions, ocular lesions and other types ofbiological samples as described herein—such as cerebrospinal fluid.

Compositions disclosed herein include amplification oligomers that canbe used to specifically amplify selected nucleic acid sequences presentin HSV genomic sequences, and nucleic acid probes for detecting theamplified sequences. Preferred embodiments include specific combinationsof oligomers to amplify and detect HSV-1 and/or HSV-2 sequences inassays that provide a detectable signal or response within about 45minutes from beginning of a transcription-associated amplificationreaction.

The disclosed nucleic acid sequences and methods are useful foramplifying and detecting HSV nucleic acids from or derived from viralparticles present in a sample in a relatively short time so thatdiagnosis can be made quickly and so effective treatment can beinitiated and spread of the virus limited. The methods are useful forscreening for individuals who have HSV infections but who do not exhibitdefinitive symptoms, or who have not seroconverted, and are particularlyuseful for screening patients who have a higher risk of death or seriouscomplications from HSV infections, e.g., young, elderly, orimmunocompromised individuals. The methods are also useful for rapidscreening of many samples. The methods are useful because they minimizethe risk of exposure of laboratory personnel to the infectious HSVagents, thereby limiting the risk of infection and spread of the virus.Thus, the methods and compositions disclosed herein respond to a needfor rapid, sensitive, and specific testing of clinical samples that maycontain HSV.

II. Definitions

To aid in understanding aspects of the disclosure, some terms usedherein are described in more detail. All other scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art, such as may be provided in Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), andreferences cited herein. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methods well-known to aperson of ordinary skill in the art of molecular biology.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly dictates otherwise. For example, “a nucleic acid,” asused herein, is understood to represent one or more nucleic acids. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

A “sample” or “specimen,” including “biological” or “clinical” samplesmay contain or may be suspected of containing HSV or components thereof,such as nucleic acids or fragments of nucleic acids. A sample may be acomplex mixture of components. Samples include “biological samples”which include any tissue or material derived from a living or deadmammal or organism, including, e.g., blood, plasma, serum, blood cells,saliva, and mucous, cerebrospinal fluid (to diagnose HSV infections ofthe central nervous system) and samples—such as biopsies—from or derivedfrom genital lesions, anogenital lesions, oral lesions, mucocutanoeuslesions, skin lesions and ocular lesions or combinations thereof.Samples may also include samples of in vitro cell culture constituentsincluding, e.g., conditioned media resulting from the growth of cellsand tissues in culture medium. The sample may be treated to physicallyor mechanically disrupt tissue or cell structure to releaseintracellular nucleic acids into a solution which may contain enzymes,buffers, salts, detergents and the like, to prepare the sample foranalysis. In one step of the methods described herein, a sample isprovided that is suspected of containing at least a HSV target nucleicacid. Accordingly, this step excludes the physical step of obtaining thesample from a subject.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5-methylisocytosine, isoguanine; TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., 2004, Biochemistry 43(42):13233-41). Nucleic acids may includemodified bases to alter the function or behavior of the nucleic acid,e.g., addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well known in the art although nucleicacids may be purified from natural sources using routine techniques.

The term “polynucleotide” denotes a nucleic acid chain. Throughout thisapplication, nucleic acids are designated by the 5′-terminus to the3′-terminus. Standard nucleic acids, e.g., DNA and RNA, are typicallysynthesized “3′-to-5′,” i.e., by the addition of nucleotides to the5′-terminus of a growing nucleic acid.

A “nucleotide” is a subunit of a nucleic acid consisting of a phosphategroup, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar foundin RNA is ribose. In DNA, the 5-carbon sugar is 2′-deoxyribose. The termalso includes analogs of such subunits, such as a methoxy group at the2′ position of the ribose (2′-O-Me, or 2′ methoxy). As used herein,methoxy oligonucleotides containing “T” residues have a methoxy group atthe 2′ position of the ribose moiety, and a uracil at the base positionof the nucleotide.

A “non-nucleotide unit” is a unit that does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” is a nucleic acid comprising a “target sequence”or “target region” to be amplified. Target nucleic acids may be DNA orRNA and may be either single-stranded or double-stranded. In a preferredembodiment of the invention, the target nucleic acid is RNA. In a morepreferred embodiment, the target sequence is RNA encoded by at least aportion of either or both of the DNA sequences set forth in FIGS. 1 and2 (illustrated as FIG. 1A, FIG. 1B, FIG. 1C, FIG. 2A, and FIG. 2B) (SEQID NOs:1 and 2). The target nucleic acid may include other sequencesbesides the target sequence that may be amplified. In the instantdisclosure, target nucleic acids are nucleic acids—such as DNA orRNA—from HSV, including HSV-1 and/or HSV-2. In a preferred embodiment,the target nucleic acid is RNA from HSV, including HSV- 1 and/or HSV-2.In another preferred embodiment, the target nucleic acid comprises RNAencoded by the DNA sequence set forth in SEQ ID NOS: 1 (HSV-1) or SEQ IDNO: 2 (HSV-2). In another preferred embodiment, the target nucleic acidis RNA from HSV that has not been obtained by reverse transcription ofHSV DNA. In other words, according to this embodiment, the targetnucleic acid is RNA obtained directly from the virus or a cell infectedwith same.

In the context of nucleic acid amplification, the term “target sequence”is used interchangeably with the term “target region” to refer to theparticular nucleotide sequence of the target nucleic acid that is to beamplified. The “target sequence” includes the complexing sequences towhich oligonucleotides (e.g., priming oligonucleotides and/or promoteroligonucleotides) stably hybridize during an amplification process(e.g., PCR, TMA). In the specific context of oligonucleotidehybridization (e.g., hybridization of an amplification oligomer ordetection probe to a segment of a target nucleic acid), the term “targetsequence” refers to the sufficiently complementary region to which theoligonucleotide (or a portion thereof) stably hybridizes. Where thetarget nucleic acid is originally single-stranded, the term “targetsequence” will also refer to the sequence complementary to the targetsequence as present in the target nucleic acid. Where the target nucleicacid is originally double-stranded, the term “target sequence” refers toboth the sense (+) and antisense (−) strands. In choosing a targetsequence, the skilled artisan will understand that a sequence should bechosen so as to distinguish between unrelated or closely related targetnucleic acids.

The terms “target(s) a sequence” or “target(s) a target nucleic acid” asused herein in reference to a region of HSV nucleic acid refers to aprocess whereby an oligonucleotide stably hybridizes to the targetsequence in a manner that allows for amplification and/or detection asdescribed herein. In one embodiment, the oligonucleotide iscomplementary to the targeted HSV nucleic acid sequence and contains nomismatches. In another embodiment, the oligonucleotide is complementarybut contains 1; or 2; or 3; or 4; or 5 mismatches with the targeted HSVnucleic acid sequence. Preferably, the oligonucleotide that stablyhybridizes to the HSV nucleic acid sequence includes at least 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45or 50 nucleotides complementary to the target sequence. It is understoodthat at least 10 and as many as 50 is an inclusive range such that 10,50 and each whole number there between are included. The term“configured to target a sequence” as used herein means that the targethybridizing region of an amplification oligonucleotide is designed tohave a polynucleotide sequence that could target a sequence of thereferenced HSV region, particularly, the referenced HSV-1 or HSV-2region. Such an amplification oligonucleotide is not limited totargeting that sequence only, but is rather useful as a composition, ina kit or in a method for targeting a HSV target nucleic acid, as isdescribed herein. The term “configured to” denotes an actual arrangementof the polynucleotide sequence configuration of the amplificationoligonucleotide target hybridizing sequence.

Oligomer target-hybridizing sequences defined herein by reference to aspecific sequence (e.g., by reference to a region within SEQ ID NO:1 orSEQ ID NO:2) are also understood to include functional complementsthereof, unless the context clearly dictates otherwise. Thus, forexample, where target-hybridizing regions of first and secondamplification oligomers are defined by reference to specific sequencescorresponding, respectively, to sense and antisense strands of a targetnucleic acid, it is understood that the amplification oligomercombination may include a functional combination of first and secondamplification oligomers having target-hybridizing sequences that are therespective complements of the specific reference sequences. Similarly,and again by way of example, where a target-hybridizing sequence for adetection probe oligomer is defined reference to a specific sequence, itis understood that the detection probe may include a correspondingdetection probe oligomer having a target-hybridizing sequence that isthe complement of the specific reference sequence; or where a detectionprobe oligomer is defined by its configuration to hybridize to aspecific sequence, it is understood that the detection probe may includea corresponding detection probe oligomer having a target-hybridizingsequence that is configured to hybridize to the complement of thespecific reference sequence. Oligomer sequences defined herein byreference to a specific sequence are also understood to include the DNAand RNA equivalents thereof (including DNA and RNA equivalents offunctional complements thereof), unless the context clearly dictatesotherwise.

The term “isolated,” in reference to a nucleic acid, means that thenucleic acid is taken from its natural milieu, but the term does notconnote any degree of purification.

The term “fragment,” as used herein in reference to an HSV targetnucleic acid, refers to a piece of contiguous nucleic acid, wherein thenumber of contiguous nucleotides in the fragment are less than that forthe entire target nucleic acid.

The term “region” refers to a portion of a nucleic acid wherein theportion is smaller than the entire nucleic acid. For example, when thenucleic acid in reference is an oligonucleotide promoter provider, theterm “region” may be used refer to the smaller promoter portion of theentire oligonucleotide. Similarly, and also as example only, when thenucleic acid is a target nucleic acid, the term “region” may be used torefer to a smaller area of the nucleic acid, wherein the smaller area istargeted by one or more oligonucleotides of the invention. For example,in reference to a target nucleic acid, “targets region” may be used torefer to a portion of the target nucleic acid to be amplified. Asanother non-limiting example, when the nucleic acid is in reference toan amplicon, the term “region” may be used to refer to the smallernucleotide sequence identified for hybridization by thetarget-hybridizing sequence of a probe.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range of from about 5 nt residues toabout 900 nt residues, from about 10 nt residues to about 800 ntresidues with a lower limit of about 12 to 15 nt and an upper limit ofabout 40 to 600 nt, and other embodiments are in a range having a lowerlimit of about 15 to 20 nt and an upper limit of about 22 to 100 nt. Itis understood that these ranges are exemplary only, and anoligonucleotide may contain each whole number included in the range.Oligonucleotides may be purified from naturally occurring sources, butmay be synthesized using any of a variety of well-known enzymatic orchemical methods. The term oligonucleotide does not denote anyparticular function to the reagent; rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions. For example, it may function as a primer ifit is specific for and capable of hybridizing to a complementary strandand can further be extended in the presence of a nucleic acidpolymerase, it may provide a promoter if it contains a sequencerecognized by an RNA polymerase and allows for transcription (e.g., a T7provider), and it may function to prevent hybridization or impede primerextension if appropriately situated and/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

As used herein, an oligonucleotide “substantially corresponding to” aspecified nucleic acid sequence means that the oligonucleotide issufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.One skilled in the art will understand that “substantially correspondingoligonucleotides” can vary from a reference sequence and still hybridizeto the same target nucleic acid sequence. It is also understood that afirst nucleic acid corresponding to a second nucleic acid includes theRNA and DNA equivalents thereof and includes the complements thereof,unless the context clearly dictates otherwise. This variation from thenucleic acid may be stated in terms of a percentage of identical baseswith the reference sequence or the percentage of perfectly complementarybases between the oligonucleotide and its target sequence. Thus, incertain embodiments, an oligonucleotide “substantially corresponds” to areference nucleic acid sequence if these percentages of base identity orcomplementarity are from 100% to about 80%. In preferred embodiments,the percentage is from 100% to about 85%. In more preferred embodiments,this percentage is from 100% to about 90%; in other preferredembodiments, this percentage is at from 100% to about 95%, to about 96%,to about 97%, to about 98% or to about 99%. One skilled in the art willunderstand that the recited ranges include all whole and rationalnumbers of the range (e.g., 92% or 92.377%). One skilled in the art willfurther understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

A “helper oligonucleotide” or “helper” refers to an oligonucleotidedesigned to bind to a target nucleic acid and impose a differentsecondary and/or tertiary structure on the target to increase the rateand extent of hybridization of a detection probe or otheroligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides. Helperoligonucleotides may be used in the methods described herein and mayform part of the compositions and kits described herein.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase.

An “amplification oligomer”, which may also be called an “amplificationoligonucleotide,” is an oligomer, at least the 3′-end of which iscomplementary to a target nucleic acid (“target hybridizing sequence”),and which hybridizes to a target nucleic acid, or its complement, andparticipates in a nucleic acid amplification reaction. An example of anamplification oligomer is a “primer” that hybridizes to a target nucleicacid and contains a 3′ OH end that is extended by a polymerase in anamplification process. Another example of an amplification oligomer is a“promoter-based amplification oligomer,” which comprises a targethybridizing sequence and a promoter sequence for initiatingtranscription by an appropriate polymerase. Promoter-based amplificationoligomers may or may not be extended by a polymerase in a primer-basedextension depending upon whether or not the 3′ end of the targethybridizing sequence is modified to prevent primer-based extension(e.g., a 3′ blocked end). A promoter-based amplification oligonucleotidecomprising a target hybridizing region that is not modified to preventprimer-based extension is referred to as a “promoter-primer.” Apromoter-based amplification oligonucleotide comprising a targethybridizing region that is modified to prevent primer-based extension isreferred to as a “promoter-provider.” Size ranges for amplificationoligonucleotides include those comprising target hybridizing regionsthat are about 10 to about 70 nt long—such as about 10 to about 60 ntlong, about 10 to about 50 nt long, about 10 to about 40 nt long, about10 to about 30 nt long or about 10 to about 25 nt long or about 15 to 25nt long. Preferred sizes of amplification oligomers include thosecomprising target hybridizing regions that are about 18, 19, 20, 21, 22or 23 nt long. An amplification oligomer may optionally include modifiednucleotides or analogs that are not complementary to target nucleic acidin a strict A:T/U, G:C sense. Such modified nucleotides or analogs areherein considered mismatched to their corresponding target sequence. Forsome embodiments, the preferred amount of amplification oligomer perreaction is about 10, 15 or 20 pmoles.

Oligomers not intended for primer-based extension by a nucleic acidpolymerase may include a blocker group that replaces the 3′OH to preventthe enzyme-mediated extension of the oligomer in an amplificationreaction. For example, blocked amplification oligomers and/or detectionprobes present during amplification may not have functional 3′OH andinstead include one or more blocking groups located at or near the 3′end. In some embodiments, a blocking group near the 3′ end is withinfive residues of the 3′ end and is sufficiently large to limit bindingof a polymerase to the oligomer. In other embodiments, a blocking groupis covalently attached to the 3′ terminus. Many different chemicalgroups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

The term “promoter,” as used herein, refers to a specific nucleic acidsequence that is recognized by a DNA-dependent RNA polymerase(“transcriptase”) as a signal to bind to the nucleic acid and begin thetranscription of RNA at a specific site.

As used herein, a “promoter-provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter-provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The target-hybridizing portion of a promoteroligonucleotide is typically at least 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 nucleotides in length,and may extend up to 50 or more nucleotides in length. The “secondregion” comprises a promoter sequence for an RNA polymerase. Apromoter-provider oligonucleotide is configured so that it is incapableof being extended by an RNA- or DNA-dependent DNA polymerase, (e.g.,reverse transcriptase), preferably by comprising a blocking moiety atits 3′-terminus as described above. This modification differentiatespromoter providers from promoter primers. Preferably, the promoterportion of a promoter primer or provider is a promoter for aDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6, though other promoters or modified version thereof can be used aswell.

As used herein, a “terminating oligonucleotide” or “blockeroligonucleotide” is an oligonucleotide comprising a base sequence thatis complementary to a region of the target nucleic acid in the vicinityof the 5′-end of the target sequence, so as to “terminate” primerextension of a nascent nucleic acid that includes a primingoligonucleotide, thereby providing a defined 3′-end for the nascentnucleic acid strand.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Known amplification methods include both thermalcycling and isothermal amplification methods. In some embodiments,isothermal amplification methods are preferred. Replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification arenon-limiting examples of nucleic acid amplification methods.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600).PCR amplification uses a DNA polymerase, pairs of primers, and thermalcycling to synthesize multiple copies of two complementary strands ofdsDNA or from a cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). LCR amplification uses four or more differentoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation(e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses a primer thatcontains a recognition site for a restriction endonuclease and anendonuclease that nicks one strand of a hemimodified DNA duplex thatincludes the target sequence, whereby amplification occurs in a seriesof primer extension and strand displacement steps (e.g., U.S. Pat. Nos.5,422,252; 5,547,861; and 5,648,211). Preferred embodiments use anamplification method suitable for the amplification of RNA targetnucleic acids, such as transcription-mediated amplification (TMA) orNASBA, but it will be apparent to persons of ordinary skill in the artthat oligomers disclosed herein may be readily used as primers in otheramplification methods.

“Transcription- associated amplification,” also referred to herein as“transcription-mediated amplification” (TMA), refers to nucleic acidamplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. These methods generally employan RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, and a template complementaryoligonucleotide that includes a promoter sequence, and optionally mayinclude one or more other oligonucleotides. TMA methods are embodimentsof amplification methods used for amplifying and detecting HSV targetsequences as described herein. Variations of transcription-associatedamplification are well-known in the art as previously disclosed indetail (e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491;5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302, WO88/10315, and WO 95/03430). The person of ordinary skill in the art willappreciate that the disclosed compositions may be used in amplificationmethods based on extension of oligomer sequences by a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “amplicon,” which is used interchangeably with “amplificationproduct,” refers to the nucleic acid molecule generated during anamplification procedure that is complementary or homologous to asequence contained within the target sequence. These terms can be usedto refer to a single strand amplification product, a double strandamplification product or one of the strands of a double strandamplification product. Double stranded amplicons can, in some aspects,be circularized using adapters. One such adapter is, for example, theSMRTBell (Pacific Biosciences, Menlo Park, Calif.). Circularized doublestranded amplicons can be useful for many purposes, including, but notlimited to, sequencing reactions.

“Probe,” “detection probe,” “detection oligonucleotide,” and “detectionprobe oligomer” are used interchangeably herein to refer to a nucleicacid oligomer that hybridizes specifically to a target sequence in anucleic acid, or in an amplified nucleic acid, under conditions thatpromote hybridization to allow detection of the target sequence oramplified nucleic acid. Detection may either be direct (e.g., a probehybridized directly to its target sequence) or indirect (e.g., a probelinked to its target via an intermediate molecular structure). Probesmay be DNA, RNA, analogs thereof or combinations thereof and they may belabeled or unlabeled. A probe's “target sequence” generally refers to asmaller nucleic acid sequence within a larger nucleic acid sequence thathybridizes specifically to at least a portion of a probe oligomer bystandard base pairing. A probe may comprise target-specific sequencesand other sequences that contribute to the three-dimensionalconformation of the probe (e.g., U.S. Pat. Nos. 5,118,801; 5,312,728;6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Pub. No.20060068417). In a preferred embodiment, the detection probe comprises a2′ methoxy backbone which can result in a higher signal being obtained.

The term “TaqMan® probe” refers to detection oligonucleotides thatcontain a fluorescent dye, typically on the 5′ base, and anon-fluorescent quenching dye (quencher), typically on the 3′ base. Whenirradiated, the excited fluorescent dye transfers energy to the nearbyquenching dye molecule rather than fluorescing, resulting in anon-fluorescent substrate. During amplification, the exonucleaseactivity of the polymerase cleaves the TaqMan probe to separate thefluorophore from the quencher, thereby allowing an unquenched signal tobe emitted from the fluorophore as an indicator of amplification.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labelling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labellingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (e.g.,U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels includechemiluminescent compounds, e.g., acridinium ester (“AE”) compounds thatinclude standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207,5,658,737, and 5,639,604). Synthesis and methods of attaching labels tonucleic acids and detecting labels are well known (e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat.Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). Morethan one label, and more than one type of label, may be present on aparticular probe, or detection may use a mixture of probes in which eachprobe is labeled with a compound that produces a detectable signal(e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

As used herein, structures referred to as “molecular torches” aredesigned to include distinct regions of self-complementarity (“theclosing domain”) which are connected by a joining region (“the targetbinding domain”) and which hybridize to one another under predeterminedhybridization assay conditions. All or part of the nucleotide sequencescomprising target closing domains may also function as target bindingdomains. Thus, target closing sequences can include, target bindingsequences, non-target binding sequences, and combinations thereof.

“Capture probe,” “capture oligonucleotide,” “target captureoligonucleotide,” and “capture probe oligomer” are used interchangeablyherein to refer to a nucleic acid oligomer that specifically hybridizesto a target sequence in a target nucleic acid by standard base pairingand joins to a binding partner on an immobilized probe to capture thetarget nucleic acid to a support. One example of a capture oligomerincludes an oligonucleotide comprising two binding regions: a targethybridizing sequence and an immobilized probe-binding region. Avariation of this example, the two regions may be present on twodifferent oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer the target hybridizing sequence is asequence that includes random or non-random poly-GU, poly-GT, or poly Usequences to bind non-specifically to a target nucleic acid and link itto an immobilized probe on a support (see, e.g., PCT Pub No. WO2008/016988). The immobilized probe binding region can be a nucleic acidsequence, referred to as a tail. Tails include a substantiallyhomopolymeric tail of about 10 to 40 nucleotides (e.g., A₁₀ to A₄₀), orof about 14 to 33 nt (e.g., T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence attached to the support particle orsupport matrix. Thus, a non-limiting example of preferred nucleic acidtails can in some embodiments include T₀₋₄A₁₀₋₄₀ sequences. Anotherexample of a capture oligomer comprises two regions, a targethybridizing sequence and a binding pair member that is not a nucleicacid sequence.

As used herein, an “immobilized oligonucleotide,” “immobilized probe” or“immobilized nucleic acid” refers to a nucleic acid binding partner thatjoins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample. Oneembodiment of an immobilized probe is an oligomer joined to a supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Supports may include known materials, such asmatrices and particles free in solution, which may be made ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal, or other compositions, of which oneembodiment is magnetically attractable particles. Supports may bemonodisperse magnetic spheres (e.g., uniform size±5%), to which animmobilized probe is joined directly (via covalent linkage, chelation,or ionic interaction), or indirectly (via one or more linkers), wherethe linkage or interaction between the probe and support is stableduring hybridization conditions.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. Sequences that hybridize to each other may becompletely complementary or partially complementary to the intendedtarget sequence by standard nucleic acid base pairing (e.g., G:C, A:T orA:U pairing). By “sufficiently complementary” is meant a contiguoussequence that is capable of hybridizing to another sequence by hydrogenbonding between a series of complementary bases, which may becomplementary at each position in the sequence by standard base pairingor may contain one or more residues that are not complementary bystandard A:T/U and G:C pairing or are modified nucleotides such asabasic residues, modified nucleotides or nucleotide analogs.Sufficiently complementary contiguous sequences typically are at least80%, or at least 90%, complementary to a sequence to which an oligomeris intended to specifically hybridize (a %-complementarity rangeincludes all whole and rational numbers of the range). Sequences thatare “sufficiently complementary” allow stable hybridization of a nucleicacid oligomer with its target sequence under appropriate hybridizationconditions, even if the sequences are not completely complementary. Whena contiguous sequence of nucleotides of one single-stranded region isable to form a series of “canonical” hydrogen-bonded base pairs with ananalogous sequence of nucleotides of the other single-stranded region,such that A is paired with U or T and C is paired with G, thenucleotides sequences are “completely” complementary (see, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at§§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

By “preferentially hybridize” or “specifically hybridize” is meant thatunder stringent hybridization assay conditions, an oligonucleotidehybridizes to its target sequences, or replicates thereof, to formstable oligonucleotide: target sequence hybrid, while at the same timeformation of stable oligonucleotide: non-target sequence hybrid isminimized. For example, a probe oligonucleotide preferentiallyhybridizes to a target sequence or replicate thereof to a sufficientlygreater extent than to a non-target sequence, to enable one havingordinary skill in the art to accurately detect the RNA replicates orcomplementary DNA (cDNA) of the target sequence formed during theamplification. Appropriate hybridization conditions are well known inthe art for probe, amplification, target capture, blocker and otheroligonucleotides, may be predicted based on sequence composition, or canbe determined by using routine testing methods (see, e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

By “nucleic acid hybrid,” “hybrid,” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

“Sample preparation” refers to any steps or methods that treat a samplefor subsequent amplification and/or detection of HSV nucleic acidspresent in the sample. The target nucleic acid may be a minoritycomponent in the sample. Sample preparation may include any known methodof isolating or concentrating components, such as viruses or nucleicacids using standard microbiology methods. Sample preparation mayinclude physical disruption and/or chemical lysis of cellular componentsto release intracellular components into a substantially aqueous ororganic phase and removal of debris, such as by using filtration,centrifugation or adsorption. Sample preparation may include use of anucleic acid oligonucleotide that selectively or non-specificallycaptures a target nucleic acid and separates it from other samplecomponents (e.g., as described in U.S. Pat. No. 6,110,678 and PCT Pub.No. WO 2008/016988).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components. Ranges of %-purity includeall whole and rational numbers of the range.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli , bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes possessing the same or similar activity as RNAse H may also beused. Selective RNAses may be endonucleases or exonucleases. Mostreverse transcriptase enzymes contain an RNAse H activity in addition totheir polymerase activities. However, other sources of the RNAse H areavailable without an associated polymerase activity. The degradation mayresult in separation of RNA from a RNA:DNA complex. Alternatively, aselective RNAse may simply cut the RNA at various locations such thatportions of the RNA melt off or permit enzymes to unwind portions of theRNA. Other enzymes that selectively degrade RNA target sequences or RNAproducts of the present invention will be readily apparent to those ofordinary skill in the art.

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms ofnucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(e.g., the signal-to-noise ratio).

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, e.g., the ratio of specific amplicons toside-products.

As used herein, the term “relative fluorescence unit” (“RFU”) is anarbitrary unit of measurement of fluorescence intensity. RFU varies withthe characteristics of the detection means used for the measurement.

III. Oligonucleotides for Amplification and Detection of HSV

Compositions disclosed herein include, e.g., amplification oligomersthat can be used to specifically amplify selected HSV-1 or HSV-2 nucleicacid sequences, and optionally nucleic acid probes for detecting theamplified sequences.

Oligonucleotides for amplifying an HSV-1 or HSV-2 target nucleic acidtypically comprise at least two amplification oligomers. Someembodiments of the invention may utilize three, four, five, six, seven,or even eight or more amplification oligomers in, for example, multiplexamplification assays. Thus, by way of example, oligonucleotides foramplifying an HSV-1 and/or HSV-2 target nucleic acid may comprise one,two, three, four, or five or more forward amplification primers and one,two, three, four, or five or more reverse amplification primers. In oneembodiment, at least two amplification oligomers are used in order togenerate an amplicon that can be subsequently detected, where the atleast two amplification oligomers are configured to specificallyhybridize to a region within a target nucleic acid selected from (a) atarget nucleic corresponding to the HSV-1 UL42 gene and (b) a targetnucleic acid corresponding to the HSV-2 UL42 gene. Suitably, theamplicon is detectable using a detection probe. Typically, the ampliconis from about 50 to about 200 nucleotides in length (e.g., about 80 toabout 140 nucleotides in length or about 90 to about 115 nucleotides inlength), including all whole numbers between 50 and 200 that are notexplicitly listed here. In particular variations, at least twoamplification oligomers for amplifying an HSV-1 target nucleic acidregion are configured to specifically hybridize to a regioncorresponding to nucleotides 602-709 or 617-709 of SEQ ID NO:1; and/orat least two amplification oligomers for amplifying an HSV-2 targetnucleic acid region are configured to specifically hybridize to a regioncorresponding to nucleotides 524-605, 581-654, or 524-654 of SEQ IDNO:2. In certain embodiments, a combination of oligonucleotides includesamplification oligomers selected from oligomers described herein foramplifying both an HSV-1 target nucleic acid and an HSV-2 target nucleicacid.

In particular embodiments of the present invention, at least twoamplification oligomers for amplifying an HSV-1 or HSV-2 target nucleicacid comprise (i) a first amplification oligomer that includes atarget-hybridizing sequence substantially corresponding to, comprising,consisting of, or consisting essentially of a target-specific oligomersequence as shown in Table 17, infra (or substantially corresponding to,comprising, consisting of, or consisting essentially of a sequencecontained in an oligomer region and including a core oligomer sequenceas shown in Table 17), and (ii) a second amplification oligomer thatincludes a target-hybridizing sequence substantially corresponding to,comprising, consisting of, or consisting essentially of atarget-specific oligomer sequence as shown in Table 17 (or substantiallycorresponding to, comprising, consisting of, or consisting essentiallyof a sequence contained in an oligomer region and including a coreoligomer sequence as shown in Table 17), where the first and secondamplification oligomers correspond to the same target nucleic acid, andwhere the target-hybridizing sequences are selected such that, for anyoligomer pair, an antisense sequence is situated downstream of a sensesequence (i.e., the first and second amplification oligomers aresituated such that they flank a target region to be amplified). Inspecific variations, the first and/or second amplification oligomer—orthe first and/or second target-hybridizing sequence of a first and/orsecond amplification oligomer—comprises or consists of an oligomersequence selected from the amplification oligonucleotide sequences shownin Table 17. The probe target-hybridizing sequences disclosed in Table17 may be used as amplification oligomer target-hybridization regions;likewise, amplification oligomer target-hybridizing sequences disclosedin Table 17 may be used as probe target-hybridizing sequences. Althoughthe oligomer sequences in Table 17 are shown as either DNA or RNA,equivalent RNA or DNA sequences, respectively, can be readily derived bythe person skilled in the art and are to be considered as falling withinthe definition of “oligomer.” In addition, complementary sequences ofDNA and RNA and reverse complementary sequences can be readily derivedby the skilled person. It is therefore to be understood that adescription of any individual sequence of DNA, for example, encompassesits complement, its reverse complement, and equivalent RNA sequences.

In certain aspects of the invention, a combination of at least twooligomers is provided for determining the presence or absence of HerpesSimplex Virus 1 (HSV-1) in a sample. Typically, the oligomer combinationincludes first and second amplification oligomers for amplifying atarget region of an HSV-1 target nucleic acid corresponding to a regionof the nucleotide sequence shown in SEQ ID NO:31. For example, in someembodiments, the first amplification oligomer comprises a firsttarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides in length and substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:31 and/orincludes at least the sequence of SEQ ID NO:30; and/or the secondamplification oligomer comprises a second target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides in length andsubstantially corresponding to, or identical to, a sequence that iscontained in the sequence of SEQ ID NO:33 and/or includes at least thesequence of SEQ ID NO:32. Typically, the first target-hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:31 and includes at leastthe sequence of SEQ ID NO:30; and the second target-hybridizing sequencesubstantially corresponds to, or is identical to, a sequence that iscontained in the sequence of SEQ ID NO:33 and includes at least thesequence of SEQ ID NO:32.

In some embodiments of the oligomer combination for determining thepresence or absence of HSV-1, the first target-hybridizing sequencesubstantially corresponds to, or is identical to, a sequence that iscontained in the sequence of SEQ ID NO:29 and/or includes at least thesequence of SEQ ID NO:28. In typical variations, the firsttarget-hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:29 and includes at least the sequence of SEQ ID NO:28. In specificvariations, the first target-hybridizing sequence substantiallycorresponds to, or is identical to, the sequence shown in SEQ ID NO:20,SEQ ID NO:6, or SEQ ID NO:12. In certain embodiments, the secondtarget-hybridizing sequence substantially corresponds to, or isidentical to, the sequence shown in SEQ ID NO:7 or SEQ ID NO:9.

In more particular variations of an oligomer combination as above, thefirst and second target-hybridizing sequences substantially correspondto, or are identical to, the sequences shown in (i) SEQ ID NO:20 and SEQID NO:7, respectively (ii) SEQ ID NO:6 and SEQ ID NO:7, respectively, or(iii) SEQ ID NO:6 and SEQ ID NO:9, respectively.

In certain embodiments, an amplification oligomer for determing thepresence or absence of HSV-1 is a promoter primer or promoter providerfurther comprising a promoter sequence located 5′ to thetarget-hybridizing sequence and which is not non-complementary to theHSV-1 target nucleic acid. For example, in some embodiments of anoligomer combination as described herein for amplification of an HSV-1UL42 target region, the first amplification oligomer is a promoterprimer or promoter provider further comprising a 5′ promoter sequence.In particular embodiments, the promoter sequence is a T7 RNA polymerasepromoter sequence such as, for example, a T7 promoter sequence havingthe nucleotide sequence shown in SEQ ID NO:54. In specific variations,the first amplification oligomer is a promoter primer or promoterprovider having the sequence shown in SEQ ID NO:19, SEQ ID NO:5, or SEQID NO:11.

In some embodiments, an oligomer combination for determining thepresence or absence of HSV-1 as above further includes at least onedetection probe oligomer configured to specifically hybridize to anHSV-1 target sequence that is amplifiable using the first and secondamplification oligomers (e.g., an HSV-1 target sequence that is flankedby the target-hybridizing sequences of the first and secondamplification oligomers). In typical variations, a detection probeoligomer for use in accordance with the present invention includes atarget-hybridizing sequence that is from about 14 to about 40nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:1 from about nucleotideposition 635 to about nucleotide position 683. For example, in somevariations, the detection probe target-hybridizing sequencesubstantially corresponds to, or is identical to, a sequence that iscontained in the sequence of SEQ ID NO:40 or SEQ ID NO:41 and/orincludes at least the sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ IDNO:38; in some such embodiments, the detection probe target-hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:40 or SEQ ID NO:41 andincludes at least the sequence of SEQ ID NO:34, SEQ ID NO:35, or SEQ IDNO:38.

In some embodiments, a detection probe target-hybridizing sequence asabove substantially corresponds to, or is identical to, a sequence thatincludes at least the sequence of SEQ ID NO:34 or SEQ ID NO:35 and thatis contained in the sequence of SEQ ID NO:36 or SEQ ID NO:37.Particularly suitable detection probe target-hybridizing sequencessubstantially correspond to, or are identical to, the sequence shown inSEQ ID NO:8 or SEQ ID NO:22; in some such variations, the first andsecond amplification oligomer target-hybridizing sequences substantiallycorrespond to, or are identical to, the sequences shown in (i) SEQ IDNO:20 and SEQ ID NO:7, respectively (ii) SEQ ID NO:6 and SEQ ID NO:7,respectively, or (iii) SEQ ID NO:6 and SEQ ID NO:9, respectively.

In other embodiments, an HSV-1 detection probe target-hybridizingsequence substantially corresponds to, or is identical to, a sequencethat includes at least the sequence of SEQ ID NO:38 and that iscontained in the sequence of SEQ ID NO:39. In specific variations, thedetection probe target-hybridizing sequence substantially correspondsto, or is identical to, the sequence shown in SEQ ID NO:10; in some suchvariations, the first and second amplification oligomertarget-hybridizing sequences substantially correspond to, or areidentical to, the sequences shown in (i) SEQ ID NO:20 and SEQ ID NO:7,(ii) SEQ ID NO:6 and SEQ ID NO:7, or (iii) SEQ ID NO:6 and SEQ ID NO:9.

In other aspects of the invention, a combination of at least twooligomers is provided for determining the presence or absence of HerpesSimplex Virus 2 (HSV-2) in a sample. Typically, the oligomer combinationincludes first and second amplification oligomers for amplifying atarget region of an HSV-2 target nucleic acid corresponding to a regionof the nucleotide sequence shown in SEQ ID NO:2. For example, in someembodiments, the first amplification oligomer comprises a firsttarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides in length and substantially corresponding to, or identicalto, a sequence that (i) is contained in the sequence of SEQ ID NO:49and/or includes at least the sequence of SEQ ID NO:48 or (ii) iscontained in the sequence of SEQ ID NO:43 and/or includes at least thesequence of SEQ ID NO:42; and the second amplification oligomercomprises a second target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides in length and substantiallycorresponding to a sequence that (i) is contained in the sequence of SEQID NO:51 and/or includes at least the sequence of SEQ ID NO:50 or (ii)is contained in the sequence of SEQ ID NO:45 and/or includes at leastthe sequence of SEQ ID NO:44.

In some embodiments of the oligomer combination for determining thepresence or absence of HSV-2 is a sample, the first target hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:49 and/or includes atleast the sequence of SEQ ID NO:48; and/or the second target hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:51 and/or includes atleast the sequence of SEQ ID NO:50. In typical variations, the firsttarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:49 and includes at least the sequence of SEQ ID NO:48; and the secondtarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:51 and includes at least the sequence of SEQ ID NO:50. In particularvariations, the first target-hybridizing sequence substantiallycorresponds to, or is identical to, the sequence shown in SEQ ID NO:24and/or the second target hybridizing sequence substantially correspondsto, or is identical to, the sequence shown in SEQ ID NO:25.

In other embodiments of the oligomer combination for determining thepresence or absence of HSV-2 is a sample, the first target hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:43 and/or includes atleast the sequence of SEQ ID NO:42; and/or the second target hybridizingsequence substantially corresponds to, or is identical to, a sequencethat is contained in the sequence of SEQ ID NO:45 and/or includes atleast the sequence of SEQ ID NO:44. In typical variations, the firsttarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:43 and includes at least the sequence of SEQ ID NO:42; and the secondtarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:45 and includes at least the sequence of SEQ ID NO:44. In particularvariations, the first target-hybridizing sequence substantiallycorresponds to, or is identical to, the sequence shown in SEQ ID NO:14and/or the second target-hybridizing sequence substantially correspondsto, or is identical to, the sequence shown in SEQ ID NO:15.

In some embodiments, an amplification oligomer for determing thepresence or absence of HSV-2 is a promoter primer or promoter providerfurther comprising a promoter sequence located 5′ to thetarget-hybridizing sequence and which is not non-complementary to theHSV-2 target nucleic acid. For example, in some embodiments of anoligomer combination as described herein for amplification of an HSV-2UL42 target region, the first amplification oligomer is a promoterprimer or promoter provider further comprising a 5′ promoter sequence.In particular embodiments, the promoter sequence is a T7 RNA polymerasepromoter sequence such as, for example, a T7 promoter sequence havingthe nucleotide sequence shown in SEQ ID NO:54. In specific variations,the first amplification oligomer is a promoter primer or promoterprovider having the sequence shown in SEQ ID NO:23 or SEQ ID NO:13.

In some embodiments, an oligomer combination for determining thepresence or absence of HSV-1 as above further includes at least onedetection probe oligomer configured to specifically hybridize to anHSV-2 target sequence that is amplifiable using the first and secondamplification oligomers (e.g., an HSV-2 target sequence that is flankedby the target-hybridizing sequences of the first and secondamplification oligomers). In certain variations—e.g., where the firsttarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:49 and includes at least the sequence of SEQ ID NO:48, and the secondtarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:51 and includes at least the sequence of SEQ ID NO:50—a detectionprobe oligomer for use in accordance with the present invention includesa target-hybridizing sequence that is from about 14 to about 25nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:2 from about nucleotideposition 608 to about nucleotide position 632. In certain variations,the detection probe target-hybridizing sequence substantiallycorresponds to, or is identical to, a sequence that is contained in thesequence of SEQ ID NO:53 and includes at least the sequence of SEQ IDNO:52. Particularly suitable detection probe target-hybridizingsequences substantially correspond to, or are identical to, the sequenceshown in SEQ ID NO:27; in some such variations, the first and secondamplification oligomer target-hybridizing sequences substantiallycorrespond to, or are identical to, the sequences shown in SEQ ID NO:24and SEQ ID NO:25, respectively.

In other embodiments of an oligomer combination for determining thepresence or absence of HSV-2 comprising a detection probe—e.g., wherethe first target hybridizing sequence substantially corresponds to, oris identical to, a sequence that is contained in the sequence of SEQ IDNO:43 and includes at least the sequence of SEQ ID NO:42, and the secondtarget hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:45 and includes at least the sequence of SEQ ID NO:44—the detectionprobe oligomer includes a target-hybridizing sequence that is from about14 to about 30 nucleotides in length and configured to specificallyhybridize to a target sequence contained within SEQ ID NO:2 from aboutnucleotide position 549 to about nucleotide position 578. In certainvariations, the detection probe target-hybridizing sequencesubstantially corresponds to, or is identical to, a sequence that iscontained in the sequence of SEQ ID NO:47 and includes at least thesequence of SEQ ID NO:46. Particularly suitable detection probetarget-hybridizing sequences substantially correspond to, or areidentical to, the sequence shown in SEQ ID NO:16; in some suchvariations, the first and second amplification oligomertarget-hybridizing sequences substantially correspond to, or areidentical to, the sequences shown in SEQ ID NO:14 and SEQ ID NO:15,respectively.

Typically, a detection probe oligomer in accordance with the presentinvention further includes a label. Particularly suitable labels includecompounds that emit a detectable light signal, e.g., fluorophores orluminescent (e.g., chemiluminescent) compounds that can be detected in ahomogeneous mixture. More than one label, and more than one type oflabel, may be present on a particular probe, or detection may rely onusing a mixture of probes in which each probe is labeled with a compoundthat produces a detectable signal (see, e.g., U.S. Pat. Nos. 6,180,340and 6,350,579). Labels may be attached to a probe by various meansincluding covalent linkages, chelation, and ionic interactions, butpreferably the label is covalently attached. For example, in someembodiments, a detection probe has an attached chemiluminescent labelsuch as, e.g., an acridinium ester (AE) compound (see, e.g., U.S. Pat.Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744), which in typicalvariations is attached to the probe by a non-nucleotide linker (see,e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604, particularlyat column 10, line 6 to column 11, line 3, and Example 8; eachincorporated by reference herein). In other embodiments , a detectionprobe comprises both a fluorescent label and a quencher, a combinationthat is particularly useful in fluorescence resonance energy transfer(FRET) assays. Specific variations of such detection probes include,e.g., a TaqMan® detection probe (Roche Molecular Diagnostics) and a“molecular beacon” (see, e.g., Tyagi et al., Nature Biotechnol.16:49-53, 1998; U.S. Pat. Nos. 5,118,801 and 5,312,728).

A detection probe oligomer in accordance with the present invention mayfurther include a non-target-hybridizing sequence. Specific embodimentsof such detection probes include, for example, probes that formconformations held by intramolecular hybridization, such asconformations generally referred to as hairpins. Particularly suitablehairpin probes include a “molecular torch” (see, e.g., US Patent Nos.6,849,412; 6,835,542; 6,534,274; and 6,361,945) and a “molecular beacon”(see, e.g., Tyagi et al., supra; U.S. Pat. No. 5,118,801 and U.S.5,312,728, supra). Methods for using such hairpin probes are well-knownin the art.

In yet other embodiments, a detection probe is a linear oligomer thatdoes not substantially form a conformation held by intramolecular bonds.In specific variations, a linear detection probe oligomer includes achemiluminescent compound as the label, preferably an acridinium ester(AE) compound.

An oligomer combination for determining the presence or absence of HSV-1or HSV-2 as above may also include at least one capture probe oligomer.The capture probe oligomer typically comprises a target-hybridizingsequence substantially corresponding to a sequence contained in thecomplement of SEQ ID NO:1 or SEQ ID NO:2. In some such embodiments, thecapture probe target-hybridizing sequence is covalently attached to asequence or moiety that binds to an immobilized probe. Suitabletarget-hybridizing sequences substantially correspond to, or areidentical to, the sequence shown in SEQ ID NO:4, SEQ ID NO:18, SEQ IDNO:70, or SEQ ID NO:72. In more particular variations, the capture probeoligomer has a sequence selected from SEQ ID NO:3, SEQ ID NO:17, SEQ IDNO:69, and SEQ ID NO:71.

In certain embodiments of an oligomer combination that includes at leastone capture probe oliogmer, the capture probe oligomer comprises atarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence that is from about 15 to about 30 contiguous nucleotidescontained in the sequence of SEQ ID NO:76 and includes at least thesequence of SEQ ID NO:75. In some variations, the capture probetarget-hybridizing sequence substantially corresponds to, or isidentical to, a sequence that is contained in the sequence of SEQ IDNO:74 and/or includes at least the sequence of SEQ ID NO:75. Forexample, the target-hybridizing sequence may substantially correspondto, or be identical to, the sequence shown in SEQ ID NO:70 or SEQ IDNO:72. Typically, the capture probe target-hybridizing sequence iscovalently attached to a sequence or moiety that binds to an immobilizedprobe. In some embodiments, the oligomer combination further includes asecond capture probe oligomer comprising a target-hybridizing sequence(typically covalently attached to a sequence or moiety that binds to animmobilized probe) configured to specifically hybridize to the HSV-1and/or HSV-2 target nucleic acid. In some such variations, the secondcapture probe oligomer has a target-hybridizing sequence substantiallycorresponding to, or identical to, the sequence shown in SEQ ID NO:4 orSEQ ID NO:18. In particular variations, the first capture probe oligomerhas the sequence shown in SEQ ID NO:69 or SEQ ID NO:71 and the secondcapture probe oligomer has the sequence shown in SEQ ID NO:3 or SEQ IDNO:17.

Oligonucleotides for amplifying and detecting an HSV target are alsoshown in Table 17. Their preferred function is included for eachsequence, and for sequences identified as promoter primers as thepreferred function, the sequences include a 5′ T7 bacteriophage promotersequence from which a T7 RNA polymerase can initiate transcription underappropriate conditions. Those skilled in the art will appreciate thatanother 5′ promoter sequence may be substituted for the T7 promotersequence, which would then function with the appropriate RNA polymerasefor the chosen other promoter sequence, to make an equivalent promoterprimer oligonucleotide. Oligomers having the same target-specificsequences as the promoter primers but without the promoter sequence arealso shown (referred to as a “target hybridizing sequence” of therespective promoter primer) and are capable of functioning as primers inamplification systems that do not use promoter primers. Those skilled inthe art will recognize that oligomers identified as having a preferredfunction in target capture have target-specific portions and optionallyinclude tail portions (e.g. T₃A₃₀) which may be deleted or substitutedwith other sequences or binding moieties.

Embodiments of oligomers may include one or more modified residuesaffecting the backbone structure (e.g., 2′-methoxy substituted RNAgroups), or one or more LNA monomers, preferably at 5′ residues of aprimer oligomer, or may include a non-nucleotide linker to attach alabel to the oligomer. In a preferred embodiment, oligomers thatfunction as probes for RNA targets may be synthesized with 2′-methoxysubstituted RNA groups to promote more stable hybridization betweenprobe and target sequences.

Preferred embodiments of target capture oligomers include atarget-specific sequence that binds specifically to the HSV targetnucleic acid and a covalently linked “tail” sequence (e.g., To₀₋₄A₁₀₋₃₆)used in capturing the hybridization complex containing the targetnucleic acid to an immobilized sequence on a solid support. Captureoligomers may include at least one 2′ methoxy linkage. Embodiments ofcapture oligomers may include the target-specific sequence that binds toHSV nucleic acid attached to another binding moiety, e.g., abiotinylated sequence that binds specifically to immobilized avidin orstreptavidin. The tail sequence or binding moiety binds to animmobilized probe (e.g., complementary sequence or avidin) to capturethe hybridized target and separate it from other sample components byseparating the solid support from the mixture.

Amplification oligomer sequences, including promoter primer sequences,bind specifically to the target nucleic acid or its complementarysequence and may contain additional sequences that are nottarget-specific, e.g., the promoter sequence in a promoter primer. Atarget-specific sequence, with or without an attached promoter sequence,may serve as an amplification oligomer in a variety of in vitroamplification processes. Embodiments of the HSV assays may useamplification methods that require multiple cycling reactiontemperatures, such as PCR (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159), or may be substantially isothermal as in, for example,transcription associated amplification methods, such as TMA or NASBA(e.g., U.S. Pat. Nos. 5,399,491, 5,480,784, 5,824,518, 5,888,779,5,786,183, 5,437,990, 5,130,238, 4,868,105, and 5,124,246, and PCT Nos.WO 8801302 and WO 8810315). The HSV assays may use amplification systemsthat are detected during the amplification process (e.g., real timedetection) by including probes that emit distinguishable fluorescentsignals when the probe is bound to the intended target sequence madeduring the amplification process. Probes for real time detection includethose referred to as “molecular beacon” or “molecular switch” probes(e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al., U.S. Pat.Nos. 5,925,517 and 6,150,097, Tyagi et al., Giesendorf et al., 1998,Clin. Chem. 44(3):482-6) and “molecular torch” probes (e.g., U.S. Pat.Nos. 6,835,542 and 6,849,412, Becker et al.). Generally, such probesinclude a reporter dye attached to one end of the probe oligomer (e.g.,FAM™, TET™, JOE™, VIC™) and a quencher compound (e.g., TAMRA™ ornon-fluorescent quencher) attached to the other end of the probeoligomer, and signal production depends on whether the two ends withtheir attached compounds are in close proximity or separated.

Also provided by the present invention are detection probe oligomers andcapture probe oligomers as described herein.

IV. Methods for Amplification and Detection of HSV

In other aspects, the present invention provides methods for detectingan HSV-1 and/or HSV-2 target nucleic acid in a sample using an oligomercombination as described herein. Such a method generally includes (1)contacting the sample, suspected of containing HSV-1 or HSV-2, with atleast two oligomers for amplifying a target region of an HSV-1 or HSV-2target nucleic acid, where the at least two oligomers include first andsecond amplification oligomers as described above; (2) performing an invitro nucleic acid amplification reaction (e.g., atranscription-associated amplification reaction), where any HSV-1 orHSV-2 target nucleic acid present in the sample is used as a templatefor generating an amplification product; and (3) detecting the presenceor absence of the amplification product, thereby indicating the presenceor absence of HSV-1 or HSV-2 in the sample. A detection method inaccordance with the present invention typically further includes thestep of obtaining the sample to be contacted with the at least twooligomers. In certain embodiments, “obtaining” a sample to be used insteps (1)-(3) includes, for example, receiving the sample from a testingfacility or other location where one or more steps of the method areperformed, and/or retrieving the sample from a location (e.g., fromstorage or other depository) within a facility where one or more stepsof the method are performed. Detecting the amplified nucleic acid may beperformed by hybridizing the amplification product to a probe sequencesuch as, for example, a detection probe as described above. Detectionmay be at the end of the amplification reaction or may be performed inreal-time. For detection, the amplified nucleic acid may be labeled andbound to an unlabeled probe, but preferred embodiments bind a labeledprobe to the amplified nucleic acid. For real-time detection, a labeledprobe may be used that is detected in a homogeneous system. In certainvariations, the method is for determining the presence or absence ofboth the HSV-1 and HSV-2 target nucleic acids such as, for example, in amultiplex assay.

For embodiments utilizing a detection probe, the probe preferably islabeled and produces a signal detected in a homogeneous system, i.e.,without separation of bound probe from unbound probe. In somevariations, probes are labeled with an acridinium ester (AE) compoundfrom which a chemiluminescent signal is produced and detected in ahomogeneous system (substantially as described in detail in U.S. Pat.Nos. 5,283,174, 5,656,744, and 5,658,737). Other examples of probes maybe labeled with a fluorescent compound which emits a detectable signalonly when the probe is bound to its target, e.g., molecular switch,molecular beacon, or molecular torch probes.

In one embodiment, probes for the specific detection of HSV-1 sequencesare labeled differently to probes for the specific detection of HSV-2sequences. Thus, the signal that is obtained from the labeled probe willbe indicative of the presence of HSV-1 or HSV-2 or a combination thereofin the sample,

Assays for detection of HSV nucleic acid may include an internal control(IC) nucleic acid that is amplified and detected by using IC-specificprimers and probe in the same reaction mixtures used for HSV nucleicacid amplification and detection. Amplification and detection of theIC-specific sequence demonstrates that assay reagents and conditionswere properly used even when no HSV-specific signal is detected for atested sample (i.e., negative samples). The IC may be used as aninternal calibrator for the assay that provides a quantitative result.The IC may be a randomized sequence derived from a naturally occurringsource that is not HSV.

Sample Preparation

Preparation of samples for amplification and detection of HSV sequencesmay include methods of separating and/or concentrating viruses containedin a sample from other sample components. Sample preparation may includeroutine methods of disrupting samples or lysing samples to releaseintracellular contents, including HSV nucleic acids or genetic sequencescomprising the UL42 ORF. Sample preparation before amplification mayinclude an optional step of target capture to specifically ornon-specifically separate the target nucleic acids from other samplecomponents. Nonspecific target capture methods may involve selectiveprecipitation of nucleic acids from a substantially aqueous mixture,adherence of nucleic acids to a support that is washed to remove othersample components, other methods of physically separating nucleic acidsfrom a mixture that contains HSV nucleic acid and other samplecomponents.

In one embodiment, HSV target nucleic acids are selectively separatedfrom other sample components by specifically hybridizing the HSV targetnucleic acid to a capture oligomer specific for HSV to form a targetsequence:capture probe complex. The complex is separated from samplecomponents by binding the target:capture probe complex to an immobilizedprobe, and separating the target:capture probe:immobilized probe complexfrom the sample, as previously described (U.S. Pat. Nos. 6,110,678;6,280,952; and 6,534,273). Target capture may occur in a solution phasemixture that contains one or more capture oligonucleotides thathybridize specifically to target nucleic acids under hybridizingconditions, usually at a temperature higher than the Tm of the tailsequence immobilized probe sequence duplex. The target:capture probecomplex is captured by adjusting the hybridization conditions so thatthe capture probe tail hybridizes to the immobilized probe, and theentire complex on the support is then separated from other samplecomponents. The support with the attached immobilized probe:captureprobe:target sequence may be washed one or more times to further removeother sample components. Other embodiments link the immobilized probe toa particulate support, such as a paramagnetic bead, so that particleswith the attached target:capture probeimmobilized probe complex may besuspended in a washing solution and retrieved from the washing solution,by using magnetic attraction. To limit the number of handling steps, thetarget nucleic acid may be amplified by simply mixing the targetsequence in the complex on the support with amplificationoligonucleotides and proceeding with amplification steps.

Capture probes including a dT₃A3o tail portion are suitable forhybridization to a complementary immobilized sequence, whereas captureprobes without this tail portion can be used in conjunction with anotherligand that is a member of a binding pair (e.g., biotinylated DNA tobind to immobilized avidin or streptavidin). The complex of the captureprobe, its target HSV nucleic acid, and an immobilized binding partneror probe facilitate separation of the HSV nucleic acid from other samplecomponents, and optional washing steps may be used to further purify thecaptured viral nucleic acid.

Amplification of the HSV Target Region

Amplifying the HSV target region using two or more primers may beaccomplished using a variety of known nucleic acid amplificationreactions. For example, amplification may be achieved using PCRamplification (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159, Mullis et al.) to produce multiple DNA strands by usingthermocycling reactions that separate dsDNA and primers specific forportions of the separated strands to make additional dsDNA molecules byusing a DNA polymerase. Well-known variations of the basic PCR methodmay also be used, e.g., reverse-transcriptase PCR that uses RT toproduce a cDNA from an RNA template, and then the DNA is amplified byPCR cycles, or PCR coupled with real-time detection, both of which aresometimes referred to as RT-PCR (e.g., TaqMan One-Step RT-PCR kits,Applied Biosystems, Inc., Foster City, Calif.).

Preferably the amplification step uses a transcription-associatedamplification reaction, such as TMA (described in detail in, e.g., U.S.Pat. Nos. 5,399,491 and 5,554,516). A TMA-based assay produces many RNAtranscripts (amplicons) from a single copy of target nucleic acid (e.g.,RNA), and the amplicons are detected to indicate the presence of thetarget HSV in the sample. Briefly, in TMA-based assays, apromoter-primer hybridizes specifically to the target sequence andreverse transcriptase (RT) that includes RNaseH activity creates a firststrand cDNA by extension from the 3′ end of the promoter-primer anddigests the template strand. The cDNA is then bound by a second primerand a new strand of DNA is synthesized from the end of the second primerusing RT to create a double-stranded DNA (dsDNA) containing a functionalpromoter sequence. RNA polymerase specific for that promoter binds tothe promoter sequence and multiple RNA transcripts are produced, whicheach can act as a template for additional sequence replication using thesame steps used for the initial template. Thus, large amounts ofsingle-stranded amplified product are made using substantiallyisothermal reaction conditions.

Amplification methods that use TMA amplification may include thefollowing steps. Briefly, a single stranded target nucleic acid—such asRNA—containing the target sequence to be amplified is provided. A firstamplification oligomer is brought in contact with that target nucleicacid by hybridizing to the target sequence. The first amplificationoligomer may be a primer or a promoter primer. A suitable nucleic acidpolymerase then generates a nucleic acid strand amplification productthat is complementary to the target nucleic acid target sequence. In theinstances where the target nucleic acid is an RNA, the RNA is typicallydegraded leaving just the newly generated amplification product, whichis available for hybridization by a second amplification oligomer. Usinga primer as the first amplification oligomer, then the secondamplification oligomer is a promoter primer or promoter provider. Asuitable nucleic acid polymerase uses the newly generated amplificationproduct to which the promoter-based oligomer is hybridized as a primerto make a complementary strand of the unhybridized promoter sequence. Ifthe second amplification oligomer is a promoter primer, then acomplementary copy of the amplification product hybridized by the secondamplification oligomer is also generated. The now double strandedpromoter sequence of the promoter-based amplification is used by asuitable RNA polymerase to initiate transcription and make RNAtranscript amplification products. The first amplification oligomerprimer can then hybridize the transcribed amplification products and thesteps can repeat. Or, the target nucleic acid is RNA and the firstamplification oligomer is a promoter-based amplification oligomer. Here,the promoter based amplification oligomer is a promoter primer. Asuitable polymerase makes a first amplification product that iscomplementary to the RNA target sequence. The RNA target nucleic acid isdegraded and a second amplification oligomer is hybridized to theamplification product. A suitable polymerase makes a complement strand,thereby generating a double stranded promoter sequence. Transcription isinitiated and RNA is transcribed. The transcribed RNA is complementaryto the original target nucleic acid, thus the second amplificationoligomer hybridizes again and makes the transcribed RNA double stranded.The RNA is degraded and the remaining DNA strand is hybridized by thefirst amplification oligomer. The amplification steps can repeat. Whenthe target nucleic acid is DNA the first amplification oligomer is apromoter primer and the second amplification is a primer. Amplificationgenerally proceeds as described above, and as is described in the art.See e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491;5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302; WO88/10315 and WO 95/03430 describing TMA and other variations oftranscription-associated amplification. The amplified products may bedetected in real-time during amplification, or at the end of theamplification reaction. Detection may be performed by a number ofmethods. Probe-based detection methods use an oligonucleotide probecomprising a target hybridizing sequence that binds specifically to atarget sequence contained in the amplification products. Detection of asignal resulting from the bound probes indicates the presence of thetarget nucleic acid in the sample.

Nucleic Acid Detection

Detection of the nucleic acids may be accomplished by a variety ofmethods. Detection methods may use nucleic acid probes comprising atarget-hybridizing sequence that is complementary to a portion of theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481). Directly or indirectly labeledprobes that specifically associate with the amplified product provide adetectable signal that indicates the presence of the target nucleic acidin the sample. For example, if the target nucleic acid is HSV (e.g.HSV-1 or HSV-2) RNA, the amplified product will contain a sequence in orcomplementary to a HSV target sequence. A probe is configured to binddirectly or indirectly to a portion of the amplification product toindicate the presence of HSV in the tested sample.

Probes that hybridize to the amplified sequences include hairpinoligonucleotides such as Molecular Torches and linear oligonucleotidesthat substantially do not form conformations held by intramolecularbonds. Preferably, the probes include labels. Linear probe embodimentsmay include a chemiluminescent compound as the label, e.g., achemiluminescent AE compound attached to the probe sequence via a linker(substantially as described in U.S. Pat. Nos. 5,585,481 and 5,639,604,particularly at column 10, line 6 to column 11, line 3, and in Example 8therein). Examples of labelling positions are a central region of theprobe oligomer and near a region of A:T base pairing, at a 3′ or 5′terminus of the oligomer, and at or near a mismatch site with a knownsequence that is not the desired target sequence. Hairpin or linearprobes may be labeled with any of a variety of different types ofinteracting labels, where one interacting member is usually attached tothe 5′ end of the probe and the other interacting member is attached tothe 3′ end of the probe. Dye labeled probes, including dual labeledprobes, single labeled probes, AE labeled probes and the like, aregenerally known. Dual labeled probes can be labeled at one end with afluorescent label (“F”) that absorbs light of a particular wavelength orrange and emits light another emission wavelength or range and at theother end with a quencher (“Q”) that dampens, partially or completely,signal emitted from the excited F when Q is in proximity with thefluorophore. Such a probe may be referred to as labeled with afluorescent/quencher (F/Q) pair. One embodiment of a hairpin probe is a“molecular torch” that detects an amplified product to indicate whethera target sequence is present in the sample after the amplification step.A molecular torch probe comprises a target binding domain and a closingdomain, as is described above. These domains allow the molecular torchto exist in open and closed conformations, depending on whether thetorch is bound to a target. (See, e.g., U.S. Pat. Nos. 6,849,412;6,835,542; 6,534,274; and 6,361,945). Another hairpin probe embodimentis a “molecular beacon” which is generally described in, e.g., Tyagi etal., 1998, Nature Biotechnol. 16:49-53, and U.S. Pat. Nos. 5,118,801 and5,312,728. Methods for using such hairpin probes to detect the presenceof a target sequence are well-known in the art.

One method for detecting HSV sequences may use atranscription-associated amplification together with a molecular torch.The molecular torch is added before or during amplification, allowingdetection to be carried out without the addition of other reagents. Forexample, a molecular torch may be designed so that the Tm of thehybridized target binding region and closing region complex is higherthan the amplification reaction temperature, thusly designed to preventthe probe from prematurely binding to amplified target sequences. Afteran interval of amplification, the mixture is heated to open the torchregions and allow the target binding regions to hybridize to a portionof the amplification products. The solution is then cooled to close anyprobes not bound to amplified products by allowing the probe targetbinding and closing regions to hybridize, which effectively closes thelabel/quencher pair. Detection is then performed to generate and detectsignals from only the probes that are hybridized to the amplified targetsequences. For example, the mixture containing the F/Q labeled hairpinprobe is irradiated with the appropriate excitation light and theemission signal is measured. In other embodiments, the hairpin detectionprobe is designed so that the amplified products hybridize to the targetbinding region of the probe during amplification, resulting in changingthe hairpin to its open conformation during amplification, and theamplification reaction mixture is irradiated at intervals to detect theemitted signal from the open probes in real time during amplification.

Exemplary probes for the detection of HSV are disclosed in SEQ ID NOs:8,10, 16, 21, 22, 26, and 27. Advantageously, these probes can be used todiscriminate between HSV-1 and HSV-2 nucleic acids since theconfiguration of the probes utilizes a difference between the nucleicacid sequences from HSV-1 and HSV-2. A detection signal will only beobtained with an HSV-1 probe having a sequence as shown in any one ofSEQ ID NOs:8, 10, 21, and 22 when HSV-1 nucleic acid has been amplifiedand is detected. Likewise, a detection signal will only be obtained withan HSV-2 probe having a sequence as shown in any one of SEQ ID NOs:16,26, and 27 when HSV-2 nucleic acid has been amplified and is detected.

In a preferred embodiment, the probes for HSV-1 and HSV-2 are labeledwith a separately detectable label—such as a 5′ fluorophore—and so it ispossible to determine if the signal obtained is from an HSV-1 or anHSV-2 probe. In a further preferred embodiment, each of the probes forHSV-1 and each of the probes for HSV-2 are each labeled with aseparately detectable label.

Exemplary Method for the Amplification and Detection of HSV Nucleic Acid

In general, methods used to demonstrate amplification and detection ofHSV nucleic acid by using the compositions described herein involve thefollowing steps. HSV RNA is separated from other sample components byusing a method that attaches the target HSV nucleic acid to a solidsupport that is separated from other sample components. In preferredembodiments, viral RNA is separated from other sample components byusing a target-capture system that includes a target-specific captureprobe for the HSV viral analyte (e.g., using methods steps described inU.S. Pat. Nos. 6,110,678, 6,280,952 and 6,534,273), or a non-specificmethod for separation of nucleic acids was used (U.S. Pat. No.5,234,809). Non-specific separation of viral RNA from other samplecomponents is performed by adhering nucleic acids reversibly to a solidsupport, followed by washing and elution of the adhered nucleic acidsinto a substantially aqueous solution (e.g., using a QIAAMP™ Viral RNAMini kit, Qiagen Inc.). Isolated HSV nucleic acid is amplified forspecific target sequences contained in the genome by using TMAamplification, and the amplification products are detected aftercompletion of the amplification reaction. Signal can be detected byusing a system that incubates the reactions and detects fluorescence atdifferent wavelengths (e.g., using a DNA Engine OPTICONTM 2 system orCHROMO4™ Real-Time PCR Detector, Bio-Rad Laboratories, Inc., Hercules,Calif.).

Real-time TMA-based assays may also be used. These assays are typicallyperformed in reaction mixture that contains the analyte nucleic acid,amplification reagent (e.g., APTIMA™ reagent, Gen-Probe Incorporated,San Diego, Calif.), a T7 promoter primer (e.g., about 9 pmol/reaction),a second primer without a promoter (e.g., about 15 pmol/reaction), and adetection probe (e.g., about 0.2-0.3 pmol/reaction) for amplicondetection, in a 40 .micro.1 reaction (in a well of a standard 96-wellplate, covered with a layer of inert oil or sealing device to preventevaporation). The mixture of target nucleic acid, primers, and probe maybe incubated at about 60.deg.C for about 10 min, cooled to about42.deg.C for about 5 min, and then enzyme reagent containing RT and T7RNA polymerase is added, the mixture is mixed (e.g., 30 sec vortex) andthen incubated at about 42.deg.C for about 75-100 min for isothermalamplification during which detection of fluorescence is performed eitherduring the reaction (e.g., every 3 seconds) or at the end of thereaction. Amplification and detection steps may be performed using anincubation and open channel fluorimeter (e.g., CHROMO4™, Bio-RadLaboratories, Inc.) for real-time two-color fluorescence detection. Theassays may include an IC, as described above, i.e., a reaction mixtureincluded primers and probe for the target HSV nucleic acid andIC-specific primers and probe, each probe labeled with a separatelydetectable 5′ fluorophore. Real-time fluorescence signals are analyzedand a detection signal (time of emergence) is calculated. Time ofemergence is calculated, e.g., by using a method that analyzes thedetected signals (relative fluorescence units or RFU) relative to thesignal detection times (RFU(t) data points) to determine a time ofemergence (“T-time”), which is the time at which a RFU(t) data pointreaches a predefined threshold value (described in detail in U.S.application 60/659,874, Scalese et al., filed Mar. 10, 2005; and U.S.published application US2007-0243600). Briefly, RFU(t) data is treatedto subtract background signal (“noise” level) and curves (RFU vs time)is normalized to optimize curve fit for data between predeterminedminimum and maximum points. In general, samples that contain a higheranalyte concentration result in a steeper curve slope and an earliertime of emergence. Average times of emergence are compared to determinethe relative efficiencies of the different assay conditions, e.g., tocompare for a single known amount of analyte, the time of emergencedetected by using a PCR-based assay compared to using a TMA-based assay.

Correlation of Detection of a Target Sequence with Diagnosis

The detection of amplified target sequences characteristic of HSV-1 in abiological sample from an individual is indicative of infection byHSV-1. Detection of amplified target sequences characteristic of HSV-2in a biological sample from an individual is indicative of infection byHSV-2. Detection of both targets in the same sample is indicative ofinfection by both HSV-1 and HSV-2.

V. Reaction Mixtures and Kits

Also provided by the subject invention is a reaction mixture foramplification and/or detection of an HSV-1 and/or HSV-2 target nucleicacid. A reaction mixture in accordance with the present invention atleast comprises one or more of the following: one or more oligomercombination(s) as described herein for amplification of an HSV-1 and/orHSV-2 target nucleic acid; and one or more detection probe oligomer(s)as described herein for determining the presence or absence of HSV-1and/or HSV-2 amplification product(s). The reaction mixture may furtherinclude a number of optional components such as, for example, arrays ofcapture probe nucleic acids. For an amplification reaction mixture, thereaction mixture will typically include other reagents suitable forperforming in vitro amplification such as, e.g., buffers, saltsolutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP,dTTP, ATP, CTP, GTP and UTP), and/or enzyme(s) (e.g., DNA polymerase,reverse transcriptase, RNA polymerase), and may include test samplecomponents, in which an HSV-1 and/or HSV-2 target nucleic acid may ormay not be present. In addition, for a reaction mixture that includes adetection probe together with an amplification oligomer combination,selection of amplification oligomers and detection probe oligomers for areaction mixture are linked by a common target region (i.e., thereaction mixture will include a probe that binds to a sequenceamplifiable by an amplification oligomer combination of the reactionmixture).

Also provided by the subject invention are kits for practicing themethods as described herein. A kit in accordance with the presentinvention at least comprises one or more of the following: one or moreoligomer combination(s) as described herein for amplification of anHSV-1 and/or HSV-2 target nucleic acid; and one or more detection probeoligomer(s) as described herein for determining the presence or absenceof HSV-1 and/or HSV-2 amplification product(s). The kits may furtherinclude a number of optional components such as, for example, arrays ofcapture probe nucleic acids. Other reagents that may be present in thekits include reagents suitable for performing in vitro amplificationsuch as, e.g., buffers, salt solutions, appropriate nucleotidetriphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP),and/or enzyme(s) (e.g., DNA polymerase, reverse transcriptase, RNApolymerase). Oligomers as described herein may be packaged in a varietyof different embodiments, and those skilled in the art will appreciatethat the invention embraces many different kit configurations. Forexample, a kit may include amplification oligomers for only one of anHSV-1 and HSV-2 target region, or it may include amplification oligomersfor both HSV-1 and HSV-2 target regions. In addition, for a kit thatincludes a detection probe together with an amplification oligomercombination, selection of amplification oligomers and detection probeoligomers for a kit are linked by a common target region (i.e., the kitwill include a probe that binds to a sequence amplifiable by anamplification oligomer combination of the kit). Such a kit may comprisecontainers, each with one or more of the various oligomers optionallytogether with one or more of the reagents (e.g., enzymes) required toperform the methods described herein. The components of the kit may besupplied in concentrated form. A set of instructions for using thecomponents of the kit will also typically be included, where theinstructions may be associated with a package insert and/or thepackaging of the kit or the components thereof. Where the kit comprisescombinations of oligomers then the individual oligomers may be providedin individual form, with appropriate instructions for mixing same, orcombinations thereof that are ready mixed.

EXAMPLES Example 1 Reagents for TMA-based Assays

Unless otherwise specified, reagents commonly used in the TMA-basedassays described herein include the following. Sample transport reagent:110 mM lithium lauryl sulfate (LLS), 15 mM NaH2PO4, 15 mM Na2HPO4, 1mMEDTA, 1 mM EGTA, pH 6.7. Lysis buffer: 790 mM HEPES, 230 mM succinicacid, 10% (w/v) LLS, and 680 mM LiOH monohydrate. Target Capture Reagent(TCR): 250 mM HEPES, 1.88 M LiCl, 310 mM LiOH, 100 mM EDTA, pH 6.4, and250.micro.g/ml of paramagnetic particles (0.7-1.05 micron particles,Sera-Mag™ MG-CM) with (dT)₁₄ oligomers covalently bound thereto. WashSolution: 10 mM HEPES, 150 mM NaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v)ethanol, 0.02% (w/v) methylparaben, 0.01% (w/v) propylparaben, and 0.1%(w/v) sodium lauryl sulfate, pH 7.5. Amplification reagent: aconcentrated solution containing 125 mM HEPES, 26.7 mM rATP, 33.3 mMrGTP, 5 mM each of rCTP and UTP, 1.33 mM each of dATP, dCTP, dGTP anddTTP, 8% (w/v) trehalose, pH 7.7, to which primers and probes may beadded. TMA Enzymes: per reaction about 90 U/.micro.1 of MMLV reversetranscriptase (RT) and about 20 U/.micro.1 of T7 RNA polymerase perreaction (where 1 U of RT incorporates 1 nmol of dTTP in 10 min at37.deg.C using 200-400 .micro.M oligo-dT-primed polyA-template, and 1 Uof T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hr at37.deg.C using a T7 promoter in DNA template). Probe Reagent forAE-labeled probes: a solution of (a) 100 mM Li-succinate, 3% (w/v) LLS,10 mM mercaptoethanesulfonate (MES), and 3% (w/v) polyvinylpyrrolidon,or (b) 100 mM Li-succinate, 0.1% (w/v) LLS, and 10 mM MES. HybridizationReagent: (C-type) 100 mM succinic acid, 2% (w/v) LLS, 100 mM LiOH, 15 mMaldrithiol-2, 1.2 M LiCl, 20 mM EDTA, and 3.0% (v/v) ethanol, pH 4.7.Selection Reagent: 600 mM boric acid, 182.5 mM NaOH, 1% (v/v) octoxynol(TRITON® X-100), pH 8.5 to 9.2, to hydrolyze AE labels on unboundoligomers. Detection Reagents for AE labels are Detect Reagent I: 1 mMnitric acid and 32 mM H₂O₂, and Detect Reagent II: 1.5 M NaOH (see U.S.Pat. Nos. 5,283,174, 5,656,744, and 5,658,737).

Example 2 Sensitivity and Specificity Testing of Oligomer Combinationsfor Amplification and Detection of HSV-1 and HSV-2 Target Nucleic Acid

Analytical sensitivity testing was evaluated using in vitro RNAtranscript (IVT) synthesized from cloned HSV1 and 2 Us8.5 and UL42 RNAs.Target IVTs were spiked into APTIMA Specimen Transport Medium (STM,Gen-Probe Incorporated, USA) to concentrations of 3, 10, 30, 100, 300and 1000 copies per reaction (cpr). For each monoplex assay, 15replicates of each concentration were tested on the TIGRIS® DTS® System(Gen-Probe Incorporated). A Probit analysis was performed to determinethe 95% and 50% detection levels for each of the monoplex assays. Thedifferent combinations of amplification oligomers and detection probestested in this experiment are shown in Table 10.

TABLE 1 Different combinations of amplification oligomers and detectionprobes tested in Example 2 Condition Primer Promoter primer Detectionprobe 5 SEQ ID NO: 56 SEQ ID NO: 55 SEQ ID NO: 57 6 SEQ ID NO: 56 SEQ IDNO: 58 SEQ ID NO: 59 7 SEQ ID NO: 60 SEQ ID NO: 55 SEQ ID NO: 59 8 SEQID NO: 62 SEQ ID NO: 61 SEQ ID NO: 63 9 SEQ ID NO: 64 SEQ ID NO: 61 SEQID NO: 65 10 SEQ ID NO: 62 SEQ ID NO: 66 SEQ ID NO: 65 11 SEQ ID NO: 7SEQ ID NO: 5 SEQ ID NO: 8 12 SEQ ID NO: 9 SEQ ID NO: 5 SEQ ID NO: 10 13SEQ ID NO: 7 SEQ ID NO: 11 SEQ ID NO: 8 14 SEQ ID NO: 15 SEQ ID NO: 13SEQ ID NO: 16

The assays used forward primers (9 pmol/reaction), reverse promoterprimers (15 pmol/reaction) and a chemiluminescent acridinium esterlabeled detection probe (0.32 pmol/reaction) in a TMA reaction performedsubstantially as described above.

Exemplary results from the tests are shown in Tables 2 to 5 and areexpressed as RLU for each of the conditions tested.

TABLE 2 HSV-1 US8.5 and condition 5 1000 0 copies 3 copies 10 copies 30copies 100 copies 300 copies copies number 0 5 4 8 15 15 15 positivepercent 0 33 27 53 100 100 100 positive AVG RLU 2,400 465,933 358,867920,600 1,770,200 1,792,800 1,854,800

TABLE 3 HSV-2 US8.5 and condition 8 1000 0 copies 3 copies 10 copies 30copies 100 copies 300 copies copies Number 0 4 10 14 15 15 15 positivePercent 0 27 67 93 100 100 100 positivity AVG RLU 1,733 91,867 305,333743,200 1,342,200 1,545,133 1,551,067

TABLE 4 HSV-1 UL42 and condition 11 1000 0 copies 3 copies 10 copies 30copies 100 copies 300 copies copies Number 0 1 2 11 13 15 15 positivePercent 0 7 13 73 87 100 100 positivity AVG RLU 2,293 15,085 21,756735,712 839,977 1,219,166 1,348,715

TABLE 5 HSV-2 UL42 and condition 14 1000 0 copies 3 copies 10 copies 30copies 100 copies 300 copies copies number 0 0 6 12 15 15 15 positivepercent 0 0 40 80 100 100 100 positivity AVG RLU 16,447 16,501 356,355519,721 897,714 1,020,993 1,029,458

These data show that conditions 5, 8, 11 and 14 are capable of detectingHSV-1 and HSV-2 target nucleic acids. Table 6 further illustrates usingconditions 5 and 8 that there is no crossreactivity with these oligomerscombinations to non-target HSV US8.5. Reactions were set up as isgenerally shown above using the condition 5 and condition 8 oligomercombinations. 1×10.sup.8 copies of HSV-1 US8.5 IVT was spiked into STMand amplification and detection was performed using condition 8oligomers (HSV-2 US8.5). Likewise, 1 x 10.sup.8 copies of HSV-2 US8.5IVT was spiked into STM and amplification and detection reaction wasperformed using condition 5 oligomers (HSV-1 US8.5).

TABLE 6 Crossreactivity Crossreactivity Crossreactivity HSV-1 US8.5 IVTHSV-2 US8.5 IVT HSV-2 Condition 8 HSV-1 Condition 5 number positive 0 0percent positivity 0 0 AVG RLU 4,000 3,133

Example 3 Clinical Sensitivity of a TMA Assay for detecting HSV-1 andHSV-2 Target Nucleic Acids

Clinical sensitivity and specificity of a TMA assays was evaluated bytesting anogenital swab samples (n=406, Wishard Memorial Hospital)collected in viral transport medium (VTM). TMA detection results werecompared to ELVIS culture reference standard. VTM-based samples werediluted 1/20 in STM and tested on the TIGRIS® DTS® System (Gen-ProbeIncorporated, USA). Samples yielding negative TMA results were retestedat lower dilutions. For both US8.5 and UL42 RNA targets, combinedresults of the HSV-1 and HSV-2 TMA assays were compared to ELVIS cultureresults as the reference standard to calculate sensitivity andspecificity. PCR-sequencing assays targeting HSV1/2 Us8.5 and UL42 DNAwere used to resolve samples yielding discordant results between ELVISand the TMA assays. PCR reactions were generally prepared as 50microliter reactions containing 1X ABI PCR buffer (Life Technologies,Inc., USA), 1.5 mM magnesium, 0.4 microM of dNTPs and each oligomer,DMSO, polymerase, 5 microliters of sample in STM and water to 50microliters total volume. For the PCR assays, amplification productswere then run on an agarose gel, the bands were excised and each excisedband was separately sequenced in a sequencing reaction do identify theamplified target nucleic acid. Oligomer combinations are presented inTable 7.

TABLE 7 Different combinations of amplification oligomers and detectionprobes tested in Example 3 Condition Primer Promoter primer Detectionprobe 5 SEQ ID NO: 56 SEQ ID NO: 55 SEQ ID NO: 57 8 SEQ ID NO: 62 SEQ IDNO: 61 SEQ ID NO: 63 11 SEQ ID NO: 7 SEQ ID NO: 5 SEQ ID NO: 8 14 SEQ IDNO: 15 SEQ ID NO: 13 SEQ ID NO: 16 15 SEQ ID NO: 67 SEQ ID NO: 68* None**condition 15 is a PCR reaction. Thus, SEQ ID NO: 68 is a primer withouta promoter sequence and there was no probe in this PCR reaction.

HSV TMA assay analytical sensitivity for HSV viral RNAs showed 95% limitof detection results as follows: HSV-1 US8.5, 125 copies per reaction:HSV-2 US8.5, 63 copies per reaction; HSV-1 UL42, 71 copies per reaction;and HSV-2 UL42, 17 copies per reaction. For 50% limit of detectionresults were 16, 14, 7 and 5 copies per reaction, respectively. US8.5and UL42 targets were also evaluated for TMA inter-assay agreement,which for HSV-1 UL42 and HSV-1 US8.5 the overall agreement was 98.5%(95%CI: 96.8-99.3), and for HSV-2 UL42 and HSV-2 US8.5 the overallagreement was 99.0% (95%CI: 97.5-99.6). TMA assay results were evaluatedfor assay performance relative to ELVIS Culture results, which forcombined HSV-1/2 UL42 the results data is illustrated in Table 8 and forcombined HSV-1/2 US8.5 the results data is illustrated in Table 9.Discordant TMA/ELVIS results were resolved using PCR (result data inTable 10).

TABLE 8 Clinical performance of combined HSV1/2 UL42 TMA Assays HSV1/2UL42 ELVIS Culture Result TMA Assays Pos Neg Total Pos 180 32 212 Neg 4190 194 Total 184 222 406 Sensitivity (95% CI) 97.8% 94.5-99.2Specificity (95% CI) 85.6% 80.4-89.6

TABLE 9 Clinical performance of combined HSV1/2 Us8.5 TMA Assays HSV1/2Us8.5 ELVIS Culture Result TMA Assays Pos Neg Total Pos 181 34 215 Neg 3188 191 Total 184 222 406 Sensitivity (95% CI) 98.4% (95.3-99.4)Specificity (95% CI) 84.7% (79.4-88.8)

TABLE 10 Resolution of TMA Assay/ELVIS Culture Discordant ResultsOriginal Test # tested by TMA Assay Results PCR PCR+ PCR− HSV1/2 Us8.5TMA+/ELVIS− 30 11 19 (n = 34) TMA−/ELVIS+ 3 0 3 (n = 3)

These data show that UL42 and US8.5 TMA assays have excellent overallagreement for detection of Herpes simplex virus 1 and 2 genitalinfections. These data further show that TMA assays for HSV1 and 2 viralRNAs are more sensitive than ELVIS culture and may be more sensitivethan PCR for detection of HSV1 and 2 infections.

Example 4 Triplex Amplification and Real-Time Detection Reaction forIdentifying HSV-1 and/or HSV-2 in Clinical Specimen

Three hundred HSV clinical samples were obtained from Wishard HealthServices (Indianapolis, Indiana), each sample being identified asculture positive or culture negative. The clinical samples were receivedin Remel M4RT liquid medium (cat # R12505, Thermo Scientific, Lenexa,Kans.) or an equivalent viral culture medium, were then diluted 1:20 (2of the 300 were diluted 1:40) in sample transport reagent and thentested in a triplex (HSV-1, HSV-2 and internal control) amplificationand real-time detection assay (n=300). Internal control was a non-HSVnucleic acid target. Positive controls were in vitro transcriptsexpressed from a vector containing an HSV-1 or HSV-2 target regionobtained from a clinical specimen shown by ELVIS to be HSV-1 or HSV-2positive, respectively. Samples with an RFU range value >1000 and acurve fit slope of 0.015 or greater were identified as HSV positivesamples (U.S. App No. 2006/0276972).

The triplex amplification and real-time detection reactions wereperformed as TMA reaction using a Panther instrument Platform (Gen-ProbeIncorporated, San Diego, Calif.), wherein each amplification anddetection reaction was configured to amplify and detect HSV-1, HSV-2 anda nucleic acid internal control. Target capture was performed usingtarget capture reagent containing target capture oligomers (SEQ IDNO:17, for capture of HSV-1 and -2 and a target capture oligomer forcapture of the internal control—not shown), and containing a separate T7primer for each of HSV-1, HSV-2 and the internal control (SEQ ID NO:19for HSV-1, SEQ ID NO:23 for HSV-2, T7 primer for the internal control).The amplification reagent was divided into two separate reagents; thefirst amplification reagent containing the non-T7 primers; and thesecond amplification reagent containing the same T7 primers as thetarget capture reagent and containing detection probes. Non-T7 primerswere SEQ ID NO:7 for HSV-1, SEQ ID NO:25 for HSV-2 and a primer for theinternal control. Detection probes were configured as torches (SEQ IDNO:21 for HSV-1, SEQ ID NO:26 for HSV-2 and an internal control, alldifferently labeled for distinction during a detection reaction (e.g.,FAM, ROX, HEX)).

Target capture was performed generally described for the APTIMA Combo 2Assay on PANTHER (Gen-Probe Incorporated, Cat #302923). Briefly, lysedtarget is combined with target capture oligomers, magnetic beads joinedto immobilized probes, and T7 primers. Reaction conditions were providedso to hybridize the target capture oligomers and the T7 primers to theirintended targets, and to hybridize the target capture oligomer to animmobilized probe. A series of wash steps were performed to removecellular components, culture and transport medium and the like.Following the wash step, a first amplification reagent was added to thewashed and captured taregt nucleic acids. The first amplificationreagent contained only non-T7 primers. There was no primer-annealingstep prior to enzyme addition since the T7 primers were already bound tothe target from the target capture step. Initiation of amplificationoccured at addition of enzyme and then was followed by a 5-minuteincubation step at 42° C. The second amplification reagent containing T7primers and torches as detection probes was added to the firstamplification reagent after a 5-minute 42.deg.C incubation reaction.Real-time detection occurred during this step measured by 3 separatefluorometers used to detect FAM, ROX, and HEX labels on each of thedifferent detection probes. Reaction readings occurred for 53 minutes.Agreement between this HSV assay and ELVIS culture results is shownbelow in Table 11.

TABLE 11 Agreement of HSV Triplex Amplification and Real- Time DetectionAssay to ELVIS Culture Results. ELVIS ELVIS % Agreement Assay AssayTotal (95% Confidence Positive Negative Samples Interval) HSV Triplex157 11 168 91.3% Positive (86.1-94.6) HSV Triplex 15 117 132 91.4%Negative (85.3-95.1) Total 172 128 300 91.3% (87.6-94.0)

Table 11 shows that there is strong agreement between these real-timeamplification and detection assay results and ELVIS culture results forthe tested clinical samples. Of the 300 samples tested, only 26discordant results were observed for an overall agreement of 91.3% (95%CI: 87.6%-94.0%). Positive agreement was 91.3% (95% CI: 86.1%-94.6%).Negative agreement was 91.4% (95% CI: 85.3%-95.1%). It is notable thatboth of the samples tested at a 1:40 dilution provided amplification anddetection results that were concordant with their corresponding ELVISresults. Strong positive agreement between the instant amplification anddetection assay results and ELVIS culture results shows that the instantassay accurately detects the presence of HSV-1 and HSV-2 when targetingHSV RNA.

Example 5 Target Capture Oligomer Evaluation

The experiment described in this example demonstrates improvedsensitivity for HSV at lower copy levels using target capture oligomerHSV UL42 878-897 TC_methoxy (SEQ ID NO:69) or HSV UL42 881-900TC_methoxy in addition to target capture oligomer HSV UL42 544TC_methoxy (SEQ ID NO:17).

The basic protocol used for running HSV Triplex kits on the PANTHERinstrument was as follows:

1. Aliquot reagents into appropriate sized containers using serologicalpipettes and L1000 or L200 if necessary. See calculations table forreagent volumes.

-   -   a. AMP1/AMP2:        -   i. 2× Midpoint AMP: Add half of the total volume of AMP1        -   ii. Nuclease free water: Add half of the total volume of            AMP1 less the total volume of oligos to be spiked            -   1. Oligos are diluted in nuclease free water. This will                create a 1× solution of midpoint AMP at the desired                concentration of oligos.    -   b. TCR (Target Capture Reagent):        -   i. TCR: Add total volume of TCR less the total volume of            oligos to be spiked and IC IVT (internal control in vitro            transcript)

2. Spike oligos using L200 or L20:

-   -   a. Thaw oligos and keep on ice when not in use.    -   b. Mix oligos thoroughly before spiking    -   c. Add required volume to appropriate reagent as determined by        desired concentrations. See calculations table for oligo        concentrations and spike volumes.

3. Spike IC IVT using L200 or L20:

-   -   a. Spike IC IVT into TCR

4. Mix reagents thoroughly

5. Reconstitute enzyme, re-label bottle(s)

6. Pull appropriate panels from −20° C. freezer and thaw in roomtemperature water bath.

-   -   Invert tubes several times when thawed.

7. Prep and Prime PANTHER instrument

8. Load reagents, samples into PANTHER racks

9. Load reagents, samples onto PANTHER and select the appropriatesequence file and rep number

10. Feed PANTHER tips and MTUs (multi-tube units) as necessary

11. Retrieve data

TABLE 12 Oligos and Their Respective Concentrations pmol/μl Oligo (50 μlType Oligo Name pmol/μg pmol/rxn rxn vol) AMP1 Non-T7 GIC 4102 nonT7169.93 2.25 0.05 Non-T7 HSV1 602 nonT7 147.40 25.00 0.5 (SEQ ID NO: 7)Non-T7 HSV2 UL42 774-791 nT7 184.11 40.00 0.8 (SEQ ID NO: 25)AMP2/Promoter T7 T7 GIC (−) 4203 66.08 3.75 0.15 Torch TRU TMA GIC4180-4197 104.91 11.25 0.45 torch (−) 5A3R T7 HSV1 UL42 688-709 T7 66.4425.00 1 (SEQ ID NO: 19) Torch HSV1 UL42 664au C9(19, 20) N/A 11.00 0.445H3D 6st(+) (SEQ ID NO: 21) T7 HSV2 UL42 829-847 T7 70.27 40.00 1.6 (SEQID NO: 23) Torch HSV2 UL42 803-822 104.88 11.00 0.44 C9(20, 21) fam 5st(SEQ ID NO: 26) TCR T7 T7 GIC (−) 4203 66.08 1.88 0.02 TCO MUIC Cap 4277dT3A30 55.1 4.00 0.04 T7 HSV1 UL42 688-709 T7 66.44 5.00 0.05 (SEQ IDNO: 19) T7 HSV2 UL42 829-847 T7 70.27 10.00 0.10 (SEQ ID NO: 23) TCO HSVUL42 544 58.7 7.00 0.07 TC_methoxy (SEQ ID NO: 17) TCO HSV UL42 878-89758.53 7.00 0.07 TC_methoxy (SEQ ID NO: 69) TCO HSV UL42 881-900 58.677.00 0.07 TC_methoxy (SEQ ID NO: 71)

Bulks of AMP1 and AMP2 were built with enough volume for 3.5 100reaction test (“100-test”) kits, according to reagent calculation sheet,using the oligos and the concentrations in Table 12 above. Each bulk wasthen split into 3 kits and was paired with a different TCR. Enough TCRwas built for 3.5 kits and spiked with all common oligos and IC IVT (invitro transcript). TCR was then split into 3 25 ml aliquots and 2 werespiked with additional TCOs (see Table 13 below). IC IVT was spiked at5×10³ cpr. Experiment was run on PP115.

TABLE 13 TCR Conditions pmol/ pmol/ Kit Oligo Name rxn Oligo Name rxnKit 1 HSV UL42 544 7.00 (Control) TC_methoxy (SEQ ID NO: 17) Kit 2 HSVUL42 544 7.00 HSV UL42 878-897 7.00 (Condition 1) TC_methoxy TC_methoxy(SEQ ID NO: 17) (SEQ ID N0: 69) Kit 3 HSV UL42 544 7.00 HSVUL42 881-9007.00 (Condition 2) TC_methoxy TC_methoxy (SEQ ID NO: 17) (SEQ ID NO: 71)

Because this experiment was performed to confirm results obtained inprevious experiments and provide additional information, a higher numberof replicates was tested at lower copy levels. This experiment was alsointended to focus on HSV2 sensitivity, so more HSV2 replicates weretested than HSV1.

TABLE 14 Replicates Tested Per Analyte Copy level (cpr of IVT in STM)HSV1 replicates HSV2 replicates 0 0 5 10 10 30 30 10 30 100 5 10

Results of the assay are summarized below in Tables 15 and 16.

TABLE 15 Results for FAM Channel (HSV2) Avg Avg Row # of % Pos when AvgTtime Avg Tslope RFURange RFURange Labels Reps Tslope >.02 of Pos of Posof Pos of Neg HSV Triplex: 544TC_HSV1 Panel 10 CPR 10 0% 0 0 0 430 30CPR 10 0% 0 0 0 471 100 CPR  5 0% 0 0 0 537 HSV Triplex: 544TC_HSV2Panel  0 CPR 5 0% 0 0 0 198 10 CPR 30 23% 18.59 0.0462 12786 195 30 CPR30 17% 18.55 0.0429 11618 191 100 CPR  10 100% 17.31 0.0611 12666 0 HSVTriplex: 544TC + 878TC_HSV1 Panel 10 CPR 10 0% 0.00 0.0000 0 422 30 CPR10 0% 0.00 0.0000 0 471 100 CPR  5 0% 0.00 0.0000 0 467 HSV Triplex:544TC + 878TC_HSV2 Panel  0 CPR 5 0% 0.00 0.0000 0 242 10 CPR 30 90%17.51 0.0574 12667 200 30 CPR 30 100% 15.74 0.0860 12815 0 100 CPR  10100% 14.20 0.1144 13022 0 HSV Triplex: 544TC + 881TC_HSV1 Panel 10 CPR10 0% 0.00 0.0000 0 410 30 CPR 10 0% 0.00 0.0000 0 458 100 CPR  5 0%0.00 0.0000 0 467 HSV Triplex: 544TC + 881TC_HSV2 Panel  0 CPR 5 0% 0.000.0000 0 224 10 CPR 30 77% 17.66 0.0546 12704 184 30 CPR 30 97% 15.870.0801 12858 238 100 CPR  10 100% 14.57 0.1147 12819 0

The HSV1 panel performed as expected in this channel; no positives. 10reps of the Control (544TC_me alone) at 30 cpr were invalidated due tosample volume error. However, because the 10 cpr and the 100 cprperformed as expected, it was determined that this was not significantenough to require a repeat experiment. The addition of a TCO closer tothe HSV2 AMP region significantly improves HSV2 sensitivity. 878TC_meand 881TC_me perform both increase Ttime and Tslope of positives.However, in this experiment, it was determined that 878TC_me givesbetter sensitivity at 10 and 30 CPR than does 881TC_me.

The addition of another TCO also increases the sensitivity of HSV1 atlower copy levels (See Table 16).

TABLE 16 Results for HEX Channel (HSV1) Avg Avg Row # of % Pos when AvgTtime Avg Tslope RFURange RFURange Labels Reps Tslope >.015 of Pos ofPos of Pos of Neg HSV Triplex: 544TC_HSV1 Panel 10 CPR 10 60% 20.780.0343 5735 253 30 CPR 10 90% 18.05 0.0499 6213 250 100 CPR  5 100%16.39 0.0694 6342 0 HSV Triplex: 544TC_HSV2 Panel  0 CPR 5 0% 0.000.0000 0 234 10 CPR 30 0% 0.00 0.0000 0 233 30 CPR 30 0% 0.00 0.0000 0242 100 CPR  10 0% 0.00 0.0000 0 212 HSV Triplex: 544TC + 878TC_HSV1Panel 10 CPR 10 100% 20.55 0.0389 5827 0 30 CPR 10 100% 17.97 0.06216323 0 100 CPR  5 100% 15.30 0.0946 6489 0 HSV Triplex: 544TC +878TC_HSV2 Panel  0 CPR 5 0% 0.00 0.0000 0 247 10 CPR 30 0% 0.00 0.00000 200 30 CPR 30 0% 0.00 0.0000 0 205 100 CPR  10 0% 0.00 0.0000 0 255HSV Triplex: 544TC + 881TC_HSV1 Panel 10 CPR 10 70% 19.02 0.0467 6215255 30 CPR 10 100% 18.51 0.0529 6036 0 100 CPR  5 100% 15.62 0.0862 65020 HSV Triplex: 544TC + 881TC_HSV2 Panel  0 CPR 5 0% 0 0 0 254 10 CPR 300% 0 0 0 211 30 CPR 30 0% 0 0 0 219 100 CPR  10 0% 0 0 0 221

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

SEQUENCES

TABLE 17 Exemplary Oligomer Sequences, Reference Sequences, and RegionsSEQ ID NO: Sequence 5′→3′ Description  1GenBank Accession No. GU734771.1 (GI:290766003),HSV-1 UL42 reference sequence nucleotide positions 92,815-94,534  2GenBank Accession No. Z86099.2 (GI:6572414), nucleotideHSV-2 UL42 reference sequence positions 93,769-95,181  3GTACTGGTTGGCGCGAAACATTTAAAAAAAAAAAAAAAAAA Target capture oligomerAAAAAAAAAAAA (HSV UL42)  4 GTACTGGTTGGCGCGAAACATarget hybridizing sequence of SEQ ID NO: 3  5aatttaatacgactcactatagggagaTTCCCTGGGGAACC T7 amp oligomer AGCACCACA(HSV1 UL42)  6 TTCCCTGGGGAACCAGCACCACA Target hybridizing sequence ofSEQ ID NO: 5  7 GTTCAGCGCATATGGACGACGA Non-T7 amp oligomer (HSV1 UL42) 8 AGACGCTGATGAAGCGCGAA Detection probe oligomer (HSV1 UL42)  9ACGACGACGTCCGACGGC Non-T7 amp oligomer (HSV1 UL42) 10 GGCCGTTGAGCTAGCCADetection probe oligomer (HSV1 UL42) 11aatttaatacgactcactatagggagaGGTTCCCTGGGGAA T7 amp oligomer CCAGCACCA(HSV1 UL42) 12 GGTTCCCTGGGGAACCAGCACCA Target hybridizing sequence ofSEQ ID NO: 11 13 AATTTAATACGACTCACTATAGGGAGAAACGTGGTGGGTTTT7 amp oligomer GGCGGTC (HSV2 UL42) 14 AACGTGGTGGGTTTGGCGGTCTarget hybridizing sequence of SEQ ID NO: 13 15 ACGTCCAGCTGCGCCTCACGANon-T7 amp oligomer (HSV2 UL42) 16 TCACGAAGGTGGTGAACGCDetection probe oligomer (HSV2 UL42) 17GUACUGGUUGGCGCGAAACATTTAAAAAAAAAAAAAAAAAA Target capture oligomerAAAAAAAAAAAA (HSV UL42) 18 GUACUGGUUGGCGCGAAACATarget hybridizing sequence of SEQ ID NO: 17 19aatttaatacgactcactatagggagaTCCCTGGGGAACCA T7 amp oligomer GCACCACA(HSV1 UL42) 20 TCCCTGGGGAACCAGCACCACA Target hybridizing sequence ofSEQ ID NO: 19 21 uGAUGAAGCGCGAACUGACucauca Detection probe oligomer(HSV1 UL42) 22 GAUGAAGCGCGAACUGAC Target hybridizing sequence ofSEQ ID NO: 21 23 aatttaatacgactcactatagggagaGCAGGTGCGCGCGTT7 amp oligomer TAAAC (HSV2 UL42) 24 GCAGGTGCGCGCGTTAAACTarget hybridizing sequence of SEQ ID NO: 23 25 ACGAGACCGCCAAACCCANon-T7 amp oligomer (HSV2 UL42) 26 CUCGGCCCCAACGGCAAGUUccgagDetection probe oligomer (HSV2 UL42) 27 CUCGGCCCCAACGGCAAGUUTarget hybridizing sequence of SEQ ID NO: 26 28 TCCCTGGGGAACCAGCACCAAmp oligomer core sequence 29 GGTTCCCTGGGGAACCAGCACCACAAmp oligomer region 30 TGGGGAACCAGC Amp oligomer core sequence 31GGTTCCCTGGGGAACCAGCACCACAAAGC Amp oligomer region 32 ACGACGAAmp oligomer core sequence 33 GTTCAGCGCATATGGACGACGACGTCCGACGGCAmp oligomer region 34 GATGAAGCGCGAA Probe oligomer core sequence 35GAUGAAGCGCGAA Probe oligomer core sequence 36 AGACGCTGATGAAGCGCGAACTGACProbe oligomer region 37 AGACGCUGAUGAAGCGCGAACUGAC Probe oligomer region38 GTTGAGC TAG Probe oligomer core sequence 39 GGCCGTTGAGCTAGCCAGCGProbe oligomer region 40 GGCCGTTGAGCTAGCCAGCGAGACGCTGATGAAGCGCGAACProbe oligomer region TGAC 41 GGCCGUUGAGCUAGCCAGCGAGACGCUGAUGAAGCGCGAACProbe oligomer region UGAC 42 TGGGTTTGGCGGTC Amp oligomer core sequence43 CGAACGTGGTGGGTTTGGCGGTCTCGTCC Amp oligomer region 44 AGCTGCGCCTAmp oligomer core sequence 45 CCGACGTCCAGCTGCGCCTCACGAAGCCAmp oligomer region 46 GAAGGTGGTGA Probe oligomer core sequence 47CAGCTCACGAAGGTGGTGAACGCCGTC Probe oligomer region 48 GTGCGCGCGTTAmp oligomer core sequence 49 GGTGACGCAGGTGCGCGCGTTAAACACGAmp oligomer region 50 GACCGCCAAACCCA Amp oligomer core sequence 51GGACGAGACCGCCAAACCCACCACGTTCG Amp oligomer region 52 CCCCAACGGCProbe oligomer core sequence 53 AGCUCGGCCCCAACGGCAAGUUUUCProbe oligomer region 54 AATTTAATACGACTCACTATAGGGAGAT7 promoter sequence 69 UGGGUGCUGGUGCUGGACGATTTAAAAAAAAAAAAAAAAAATarget capture oligomer AAAAAAAAAAAA (HSV UL42) 70 UGGGUGCUGGUGCUGGACGATarget hybridizing sequence of SEQ ID NO: 69 71ACCUGGGUGCUGGUGCUGGATTTAAAAAAAAAAAAAAAAAA Target capture oligomerAAAAAAAAAAAA (HSV UL42) 72 ACCUGGGUGCUGGUGCUGGATarget hybridizing sequence of SEQ ID NO: 71 73 UGGGUGCUGGUGCUGGACapture oligomer core sequence 74 ACCUGGGUGCUGGUGCUGGACGACapture oligomer region 75 GUGCUGGUGCU Capture oligomer core sequence 76AUCUGGACCUGGGUGCUGGUGCUGGACGAC Capture oligomer region

The invention claimed is:
 1. A method for determining the presence orabsence of Herpes Simplex Virus 2 (HSV-2) in a sample, said methodcomprising: (1) contacting a sample, said sample suspected of containingHSV-2, with at least two oligomers for amplifying a target region of anHSV-2 target nucleic acid, wherein the at least two amplificationoligomers comprise (a) a first amplification oligomer comprising a firsttarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides (i) contained in the sequence of SEQ ID NO:49 and thatincludes at least the sequence of SEQ ID NO:48 or (ii) contained in thesequence of SEQ ID NO:43 and that includes at least the sequence of SEQID NO:42; and (b) a second amplification oligomer comprising a secondtarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides (i) contained in the sequence of SEQ ID NO:51 and thatincludes at least the sequence of SEQ ID NO:50 or (ii) contained in thesequence of SEQ ID NO:45 and that includes at least the sequence of SEQID NO:44; (2) performing an in vitro nucleic acid amplificationreaction, wherein any HSV-2 target nucleic acid present in said sampleis used as a template for generating an amplification product; and (3)detecting the presence or absence of the amplification product, therebyindicating the presence or absence of HSV-2 in said sample.
 2. Themethod of claim 1, wherein the first target hybridizing sequence iscontained in the sequence of SEQ ID NO:49 and includes at least thesequence of SEQ ID NO:48; and the second target hybridizing sequence iscontained in the sequence of SEQ ID NO:51 and includes at least thesequence of SEQ ID NO:50.
 3. The method of claim 2, wherein the firsttarget-hybridizing sequence has the sequence of SEQ ID NO:24 and/or thesecond target hybridizing sequence has the sequence of SEQ ID NO:
 25. 4.The method of claim 2, wherein the first target hybridizing sequence iscontained in the sequence of SEQ ID NO:43 and includes at least thesequence of SEQ ID NO: 42; and the second target hybridizing sequence iscontained in the sequence of SEQ ID NO:45 and includes at least thesequence of SEQ ID NO:44.
 5. The method of claim 4, wherein the firsttarget-hybridizing sequence has the sequence of SEQ ID NO:14.
 6. Themethod of claim 4, wherein the second target hybridizing sequence hasthe sequence of SEQ ID NO:15.
 7. The method of claim 1, wherein thefirst and second target-hybridizing sequences respectively have thenucleotide sequences of (i) SEQ ID NO:24 and SEQ ID NO:25; or (ii) SEQID NO:14 and SEQ ID NO:15.
 8. The method of claim 1, wherein the firstamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the firsttarget-hybridizing sequence.
 9. The method of claim 8, wherein thepromoter sequence is a T7 promoter sequence, optionally wherein thepromoter sequence has the sequence of SEQ ID NO:54.
 10. The method ofclaim 9, wherein the first amplification oligomer has a sequenceselected from the group consisting of SEQ ID NO:23 and SEQ ID NO:13. 11.The method of claim 1, further comprising purifying the HSV-2 targetnucleic acid from other components in the sample before step (1). 12.The method of claim 11, wherein the purifying step comprises contactingthe sample with at least one capture probe oligomer comprising (i) atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe, wherein said target-hybridizingsequence comprises SEQ ID NO:4, SEQ ID NO:1, SEQ ID NO:70, or SEQ IDNO:72; or (ii) a sequence comprising SEQ ID NO:3, SEQ ID NO:17, SEQ IDNO:69, or SEQ ID NO:71; or (iii) a target-hybridizing sequencecovalently attached to a sequence or moiety that binds to an immobilizedprobe, wherein said target-hybridizing sequence is from about 15 toabout 30 contiguous nucleotides contained in the sequence of SEQ IDNO:76 and includes at least the sequence of SEQ ID NO:75; or (iv) atarget-hybridizing sequence the is contained in the sequence of SEQ IDNO:74; or (v) a target-hybridizing sequence that is contained in thesequence of SEQ ID NO:74 and includes at least the sequence of SEQ IDNO:75.
 13. The method of claim 1, wherein the detecting step (3)comprises contacting said in vitro nucleic acid amplification reactionwith a detection probe oligomer configured to specifically hybridize tothe amplification product under conditions whereby the presence orabsence of the amplification product is determined, thereby indicatingthe presence or absence of HSV-2 in said sample.
 14. The method of claim13, wherein the detection probe oligomer comprises (i) atarget-hybridizing sequence that is from about 14 to about 25nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:2 from about nucleotideposition 608 to about nucleotide position 632; or (ii) atarget-hybridizing sequence that is contained in the sequence of SEQ IDNO:53 and includes at least the sequence of SEQ ID NO:52; or (iii) atarget-hybridizing sequence consisting of SEQ ID NO:27; or (iv) atarget-hybridizing sequence that is from about 14 to about 30nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:2 from about nucleotideposition 549 to about nucleotide position 578; or (v) atarget-hybridizing sequence that is contained in the sequence of SEQ IDNO:47 and includes at least the sequence of SEQ ID NO:46; or (vi) atarget-hybridizing sequence consisting of SEQ ID NO:16.
 15. The methodof claim 14, wherein the detection probe further comprises a label,optionally wherein the label is a chemiluminescent label or afluorescent label, further optionally wherein the label is a fluorescentlabel and the detection probe further comprises a quencher.
 16. Themethod of claim 14, wherein the detection probe further comprises anon-target-hybridizing sequence.
 17. A combination of at least twooligomers for determining the presence or absence of Herpes SimplexVirus 2 (HSV-2) in a sample, said oligomer combination comprising: firstand second amplification oligomers for amplifying a target region of anHSV-2 target nucleic acid, wherein (a) the first amplification oligomercomprises a first target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides (i) contained in the sequence of SEQ IDNO:49 and that includes at least the sequence of SEQ ID NO:48 or (ii)contained in the sequence of SEQ ID NO:43 and that includes at least thesequence of SEQ ID NO:42; and (b) the second amplification oligomercomprises a second target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides (i) contained in the sequence of SEQ IDNO:51 and that includes at least the sequence of SEQ ID NO:50 or (ii)contained in the sequence of SEQ ID NO:45 and that includes at least thesequence of SEQ ID NO:44.
 18. A reaction mixture comprising thecombination of at least two oligomers as in claim
 17. 19. Thecombination of claim 17, further comprising a detection probe oligomerconfigured to specifically hybridize to an amplification productproduced by the first and second amplification oligomers in the presenceof the under conditions whereby the presence or absence of the HSV-2target nucleic acid, wherein the detection probe oligomer comprises adetectable label.
 20. The combination of claim 17, wherein the firstamplification oligomer is a promoter primer or a promoter providerfurther comprising a promoter sequence located 5′ to the firsttarget-hybridizing sequence.